Planar cell polarity signalling couples cell division and morphogenesis during neurulation

Environmental and genetic aberrations lead to neural tube closure defects (NTDs) in 1 out of every 1,000 births. Mouse and frog models for these birth defects have indicated that Van Gogh-like 2 (Vangl2, also known as Strabismus) and other components of planar cell polarity (PCP) signalling might control neurulation by promoting the convergence of neural progenitors to the midline. Here we show a novel role for PCP signalling during neurulation in zebrafish. We demonstrate that non-canonical Wnt/PCP signalling polarizes neural progenitors along the anteroposterior axis. This polarity is transiently lost during cell division in the neural keel but is re-established as daughter cells reintegrate into the neuroepithelium. Loss of zebrafish Vangl2 (in trilobite mutants) abolishes the polarization of neural keel cells, disrupts re-intercalation of daughter cells into the neuroepithelium, and results in ectopic neural progenitor accumulations and NTDs. Remarkably, blocking cell division leads to rescue of trilobite neural tube morphogenesis despite persistent defects in convergence and extension. These results reveal a function for PCP signalling in coupling cell division and morphogenesis at neurulation and indicate a previously unrecognized mechanism that might underlie NTDs.     

Neural crest regulates myogenesis through the transient activation of NOTCH

How dynamic signalling and extensive tissue rearrangements interact to generate complex patterns and shapes during embryogenesis is poorly understood. Here we characterize the signalling events taking place during early morphogenesis of chick skeletal muscles. We show that muscle progenitors present in somites require the transient activation of NOTCH signalling to undergo terminal differentiation. The NOTCH ligand Delta1 is expressed in a mosaic pattern in neural crest cells that migrate past the somites. Gain and loss of Delta1 function in neural crest modifies NOTCH signalling in somites, which results in delayed or premature myogenesis. Our results indicate that the neural crest regulates early muscle formation by a unique mechanism that relies on the migration of Delta1-expressing neural crest cells to trigger the transient activation of NOTCH signalling in selected muscle progenitors. This dynamic signalling guarantees a balanced and progressive differentiation of the muscle progenitor pool.     

Differential positioning of adherens junctions is associated with initiation of epithelial folding

During tissue morphogenesis, simple epithelial sheets undergo folding to form complex structures. The prevailing model underlying epithelial folding involves cell shape changes driven by myosin-dependent apical constriction. Here we describe an alternative mechanism that requires differential positioning of adherens junctions controlled by modulation of epithelial apical-basal polarity. Using live embryo imaging, we show that before the initiation of dorsal transverse folds during Drosophila gastrulation, adherens junctions shift basally in the initiating cells, but maintain their original subapical positioning in the neighbouring cells. Junctional positioning in the dorsal epithelium depends on the polarity proteins Bazooka and Par-1. In particular, the basal shift that occurs in the initiating cells is associated with a progressive decrease in Par-1 levels. We show that uniform reduction of the activity of Bazooka or Par-1 results in uniform apical or lateral positioning of junctions and in each case dorsal fold initiation is abolished. In addition, an increase in the Bazooka/Par-1 ratio causes formation of ectopic dorsal folds. The basal shift of junctions not only alters the apical shape of the initiating cells, but also forces the lateral membrane of the adjacent cells to bend towards the initiating cells, thereby facilitating tissue deformation. Our data thus establish a direct link between modification of epithelial polarity and initiation of epithelial folding.     

Determining the position of the cell division plane

Proper positioning of the cell division plane during mitosis is essential for determining the size and position of the two daughter cells--a critical step during development and cell differentiation. A bipolar microtubule array has been proposed to be a minimum requirement for furrow positioning in mammalian cells, with furrows forming at the site of microtubule plus-end overlap between the spindle poles. Observations in other species have suggested, however, that this may not be true. Here we show, by inducing mammalian tissue cells with monopolar spindles to enter anaphase, that furrow formation in cultured mammalian cells does not require a bipolar spindle. Unexpectedly, cytokinesis occurs at high frequency in monopolar cells. Division always occurs at a cortical position distal to the chromosomes. Analysis of microtubules during cytokinesis in cells with monopolar and bipolar spindles shows that a subpopulation of stable microtubules extends past chromosomes and binds to the cell cortex at the site of furrow formation. Our data are consistent with a model in which chromosomes supply microtubules with factors that promote microtubule stability and furrowing.     

Planar cell polarity signalling controls cell division orientation during zebrafish gastrulation

Oriented cell division is an integral part of pattern development in processes ranging from asymmetric segregation of cell-fate determinants to the shaping of tissues. Despite proposals that it has an important function in tissue elongation, the mechanisms regulating division orientation have been little studied outside of the invertebrates Caenorhabditis elegans and Drosophila melanogaster. Here, we have analysed mitotic divisions during zebrafish gastrulation using in vivo confocal imaging and found that cells in dorsal tissues preferentially divide along the animal-vegetal axis of the embryo. Establishment of this animal-vegetal polarity requires the Wnt pathway components Silberblick/Wnt11, Dishevelled and Strabismus. Our findings demonstrate an important role for non-canonical Wnt signalling in oriented cell division during zebrafish gastrulation, and indicate that oriented cell division is a driving force for axis elongation. Furthermore, we propose that non-canonical Wnt signalling has a conserved role in vertebrate axis elongation, orienting both cell intercalation and mitotic division.     

Lgl, Pins and aPKC regulate neuroblast self-renewal versus differentiation

How a cell chooses to proliferate or to differentiate is an important issue in stem cell and cancer biology. Drosophila neuroblasts undergo self-renewal with every cell division, producing another neuroblast and a differentiating daughter cell, but the mechanisms controlling the self-renewal/differentiation decision are poorly understood. Here we tested whether cell polarity genes, known to regulate embryonic neuroblast asymmetric cell division, also regulate neuroblast self-renewal. Clonal analysis in larval brains showed that pins mutant neuroblasts rapidly fail to self-renew, whereas lethal giant larvae (lgl) mutant neuroblasts generate multiple neuroblasts. Notably, lgl pins double mutant neuroblasts all divide symmetrically to self-renew, filling the brain with neuroblasts at the expense of neurons. The lgl pins neuroblasts show ectopic cortical localization of atypical protein kinase C (aPKC), and a decrease in aPKC expression reduces neuroblast numbers, suggesting that aPKC promotes neuroblast self-renewal. In support of this hypothesis, neuroblast-specific overexpression of membrane-targeted aPKC, but not a kinase-dead version, induces ectopic neuroblast self-renewal. We conclude that cortical aPKC kinase activity is a potent inducer of neuroblast self-renewal.     

WNT11 acts as a directional cue to organize the elongation of early muscle fibres

The early vertebrate skeletal muscle is a well-organized tissue in which the primitive muscle fibres, the myocytes, are all parallel and aligned along the antero-posterior axis of the embryo. How myofibres acquire their orientation during development is unknown. Here we show that during early chick myogenesis WNT11 has an essential role in the oriented elongation of the myocytes. We find that the neural tube, known to drive WNT11 expression in the medial border of somites, is necessary and sufficient to orient myocyte elongation. We then show that the specific inhibition of WNT11 function in somites leads to the disorganization of myocytes. We establish that WNT11 mediates this effect through the evolutionary conserved planar cell polarity (PCP) pathway, downstream of the WNT/beta-catenin-dependent pathway, required to initiate the myogenic program of myocytes and WNT11 expression. Finally, we demonstrate that a localized ectopic source of WNT11 can markedly change the orientation of myocytes, indicating that WNT11 acts as a directional cue in this process. All together, these data show that the sequential action of the WNT/PCP and the WNT/beta-catenin pathways is necessary for the formation of fully functional embryonic muscle fibres. This study also provides evidence that WNTs can act as instructive cues to regulate the PCP pathway in vertebrates.     

PTK7/CCK-4 is a novel regulator of planar cell polarity in vertebrates

In addition to the apical-basal polarity pathway operating in epithelial cells, a planar cell polarity (PCP) pathway establishes polarity within the plane of epithelial tissues and is conserved from Drosophila to mammals. In Drosophila, a 'core' group of PCP genes including frizzled (fz), flamingo/starry night, dishevelled (dsh), Van Gogh/strabismus and prickle, function to regulate wing hair, bristle and ommatidial polarity. In vertebrates, the PCP pathway regulates convergent extension movements and neural tube closure, as well as the orientation of stereociliary bundles of sensory hair cells in the inner ear. Here we show that a mutation in the mouse protein tyrosine kinase 7 (PTK7) gene, which encodes an evolutionarily conserved transmembrane protein with tyrosine kinase homology, disrupts neural tube closure and stereociliary bundle orientation, and shows genetic interactions with a mutation in the mouse Van Gogh homologue vangl2. We also show that PTK7 is dynamically localized during hair cell polarization, and that the Xenopus homologue of PTK7 is required for neural convergent extension and neural tube closure. These results identify PTK7 as a novel regulator of PCP in vertebrates.     

Antero-posterior tissue polarity links mesoderm convergent extension to axial patterning

Remodelling its shape, or morphogenesis, is a fundamental property of living tissue. It underlies much of embryonic development and numerous pathologies. Convergent extension (CE) of the axial mesoderm of vertebrates is an intensively studied model for morphogenetic processes that rely on cell rearrangement. It involves the intercalation of polarized cells perpendicular to the antero-posterior (AP) axis, which narrows and lengthens the tissue. Several genes have been identified that regulate cell behaviour underlying CE in zebrafish and Xenopus. Many of these are homologues of genes that control epithelial planar cell polarity in Drosophila. However, elongation of axial mesoderm must be also coordinated with the pattern of AP tissue specification to generate a normal larval morphology. At present, the long-range control that orients CE with respect to embryonic axes is not understood. Here we show that the chordamesoderm of Xenopus possesses an intrinsic AP polarity that is necessary for CE, functions in parallel to Wnt/planar cell polarity signalling, and determines the direction of tissue elongation. The mechanism that establishes AP polarity involves graded activin-like signalling and directly links mesoderm AP patterning to CE.     

MOR1 is essential for organizing cortical microtubules in plants

Microtubules orchestrate cell division and morphogenesis, but how they disassemble and reappear at different subcellular locations is unknown. Microtubule organizing centres are thought to have an important role, but in higher plants microtubules assemble in ordered configurations even though microtubule organizing centres are inconspicuous or absent. Plant cells generate highly organized microtubule arrays that coordinate mitosis, cytokinesis and expansion. Inhibiting microtubule assembly prevents chromosome separation, blocks cell division and impairs growth polarity. Microtubules are essential for the formation of cell walls, through an array of plasma-membrane-associated cortical microtubules whose control mechanisms are unknown. Using a genetic strategy to identify microtubule organizing factors in Arabidopsis thaliana, we isolated temperature-sensitive mutant alleles of the MICROTUBULE ORGANIZATION 1 (MOR1) gene. Here we show that MOR1 is the plant version of an ancient family of microtubule-associated proteins. Point mutations that substitute single amino-acid residues in an amino-terminal HEAT repeat impart reversible temperature-dependent cortical microtubule disruption, showing that MOR1 is essential for cortical microtubule organization.     

Bazooka recruits Inscuteable to orient asymmetric cell divisions in Drosophila neuroblasts

Asymmetric cell divisions can be generated by the segregation of determinants into one of the two daughter cells. In Drosophila, neuroblasts divide asymmetrically along the apical-basal axis shortly after their delamination from the neuroectodermal epithelium. Several proteins, including Numb and Miranda, segregate into the basal daughter cell and are needed for the determination of its correct cell fate. Both the apical-basal orientation of the mitotic spindle and the localization of Numb and Miranda to the basal cell cortex are directed by Inscuteable, a protein that localizes to the apical cell cortex before and during neuroblast mitosis. Here we show that the apical localizaton of Inscuteable requires Bazooka, a protein containing a PDZ domain that is essential for apical-basal polarity in epithelial cells. Bazooka localizes with Inscuteable in neuroblasts and binds to the Inscuteable localization domain in vitro and in vivo. In embryos lacking both maternal and zygotic bazooka function, Inscuteable no longer localizes asymmetrically in neuroblasts and is instead uniformly distributed in the cytoplasm. Mitotic spindles in neuroblasts are misoriented in these embryos, and the proteins Numb and Miranda fail to localize asymmetrically in metaphase. Our results suggest that direct binding to Bazooka mediates the asymmetric localization of Inscuteable and connects the asymmetric division of neuroblasts to the axis of epithelial apical-basal polarity.     

A community effect in animal development

In animal development, the first tissues to be formed include such major components as muscle, nerve cord, notochord and the eye. In the vertebrates, all of these tissues are formed by embryonic induction, a process by which some of the cells within a mass of tissue are caused to change their direction of differentiation as a result of close proximity to cells of another kind. The induced cells typically form a solid coherent mass with a distinct border between them and the remaining uninduced cells. This clean separation between induced and uninduced cells is much sharper than can readily be explained as a result of the induction process. We describe here the culture of amphibian cell and tissue recombinations in solid gels containing cytochalasin in which cell division and cell movement is inhibited during response to induction. This has revealed an effect in which the ability of a cell to respond to induction by differentiating as muscle is enhanced by, or even dependent on, other neighbouring cells differentiating in the same way at the same time. This seems to be a newly described process in animal development, termed the community effect. It helps to explain the formation of blocks of tissue from sheets of cells, and could be of widespread occurrence and significance in morphogenesis resulting from embryonic induction.     

Polo inhibits progenitor self-renewal and regulates Numb asymmetry by phosphorylating Pon

Self-renewal and differentiation are cardinal features of stem cells. Asymmetric cell division provides one fundamental mechanism by which stem cell self-renewal and differentiation are balanced. A failure of this balance could lead to diseases such as cancer. During asymmetric division of stem cells, factors controlling their self-renewal and differentiation are unequally segregated between daughter cells. Numb is one such factor that is segregated to the differentiating daughter cell during the stem-cell-like neuroblast divisions in Drosophila melanogaster, where it inhibits self-renewal. The localization and function of Numb is cell-cycle-dependent. Here we show that Polo (ref. 13), a key cell cycle regulator, the mammalian counterparts of which have been implicated as oncogenes as well as tumour suppressors, acts as a tumour suppressor in the larval brain. Supernumerary neuroblasts are produced at the expense of neurons in polo mutants. Polo directly phosphorylates Partner of Numb (Pon, ref. 16), an adaptor protein for Numb, and this phosphorylation event is important for Pon to localize Numb. In polo mutants, the asymmetric localization of Pon, Numb and atypical protein kinase C are disrupted, whereas other polarity markers are largely unaffected. Overexpression of Numb suppresses neuroblast overproliferation caused by polo mutations, suggesting that Numb has a major role in mediating this effect of Polo. Our results reveal a biochemical link between the cell cycle and the asymmetric protein localization machinery, and indicate that Polo can inhibit progenitor self-renewal by regulating the localization and function of Numb.     

Myosin-dependent junction remodelling controls planar cell intercalation and axis elongation

Shaping a developing organ or embryo relies on the spatial regulation of cell division and shape. However, morphogenesis also occurs through changes in cell-neighbourhood relationships produced by intercalation. Intercalation poses a special problem in epithelia because of the adherens junctions, which maintain the integrity of the tissue. Here we address the mechanism by which an ordered process of cell intercalation directs polarized epithelial morphogenesis during germ-band elongation, the developmental elongation of the Drosophila embryo. Intercalation progresses because junctions are spatially reorganized in the plane of the epithelium following an ordered pattern of disassembly and reassembly. The planar remodelling of junctions is not driven by external forces at the tissue boundaries but depends on local forces at cell boundaries. Myosin II is specifically enriched in disassembling junctions, and its planar polarized localization and activity are required for planar junction remodelling and cell intercalation. This simple cellular mechanism provides a general model for polarized morphogenesis in epithelial organs.     

Cdc42 regulates GSK-3beta and adenomatous polyposis coli to control cell polarity

Cell polarity is a fundamental property of all cells. In higher eukaryotes, the small GTPase Cdc42, acting through a Par6-atypical protein kinase C (aPKC) complex, is required to establish cellular asymmetry during epithelial morphogenesis, asymmetric cell division and directed cell migration. However, little is known about what lies downstream of this complex. Here we show, through the use of primary rat astrocytes in a cell migration assay, that Par6-PKCzeta interacts directly with and regulates glycogen synthase kinase-3beta (GSK-3beta) to promote polarization of the centrosome and to control the direction of cell protrusion. Cdc42-dependent phosphorylation of GSK-3beta occurs specifically at the leading edge of migrating cells, and induces the interaction of adenomatous polyposis coli (Apc) protein with the plus ends of microtubules. The association of Apc with microtubules is essential for cell polarization. We conclude that Cdc42 regulates cell polarity through the spatial regulation of GSK-3beta and Apc. This role for Apc may contribute to its tumour-suppressor activity.     

Combinational electroporation and transplantation approach to studying gene functions in avian embryos

Gene transfection is an indispensable approach for studying gene function since it provides important information on gain- and/or loss-of-function. Chick embryos are also extensively employed for studying bio- logical function since they are easily accessible and can be maintained alive after manipulation. The combination of both techniques presents a powerful approach to under- standing how genes regulate embryo development. Fur- thermore, combining these approaches with tissue transplant techniques make even more attractive for elu- cidate gene function. Electroporation, employing parallelly fashioned electrodes, has been widely used in chick embryos. However, experimenters have been frustrated by unsuccessfully transfection in some embryonic tissue of interest because the electrodes were improperly positioned.We presently demonstrated the different patterns of orga- nizing and positioning the electrodes, in combination with tissue transplantation, to efficiently and specifically trans- fect the chick embryonic head, trunk neural tube, heart tube, somites and neural crest cells with the GFP reporter gene.     

Bazooka provides an apical cue for Inscuteable localization in Drosophila neuroblasts

Asymmetric cell division generates daughter cells with different developmental fates from progenitor cells that contain localized determinants. During this division, the asymmetric localization of cell-fate determinants and the orientation of the mitotic spindle must be precisely coordinated. In Drosophila neuroblasts, inscuteable controls both spindle orientation and the asymmetric localization of the cell-fate determinants Prospero and Numb. Inscuteable itself is localized in an apical cortical crescent and thus reflects the intrinsic asymmetry of the neuroblast. Here we show that localization of Inscuteable depends on Bazooka, a protein containing three PDZ domains with overall sequence similarity to Par-3 of Caenorhabditis elegans. Bazooka and Inscuteable form a complex that also contains Staufen, a protein responsible for the asymmetric localization of prospero messenger RNA. We propose that, after delamination of the neuroblast from the neuroepithelium, Bazooka provides an asymmetric cue in the apical cytocortex that is required to anchor Inscuteable. As Bazooka is also responsible for the maintenance of apical-basal polarity in epithelial tissues, it may be the missing link between epithelial polarity and neuroblast polarity.     

Polarizing activity and retinoid synthesis in the floor plate of the neural tube

In many developing organisms the establishment of axial polarity and the patterning of cells depend on local signals that derive from restricted regions of the embryo. In vertebrate embryos, the origins of tissue polarity have been examined extensively in the developing limb. The anteroposterior pattern of the chick limb seems to be controlled by a morphogen, possibly retinoic acid, that is enriched in a region of the limb known as the zone of polarizing activity (ZPA). Certain tissues other than the ZPA have also shown polarizing activity experimentally in the chick limb, raising the possibility that signalling molecules involved in pattern formation in different embryonic tissues are conserved. Here we provide evidence that a similar polarizing activity is also present in a restricted region of the developing central nervous system (CNS). We show that a specialized group of neural cells termed the floor plate, but not other regions of the CNS, mimics the ZPA in respecifying the digit pattern in the developing chick limb. In addition, using an in vitro biochemical assay, we show that the floor plate can synthesize retinoic acid and 3,4-didehydroretinol, the precursor of a second morphogenetically active retinoid, 3,4-didehydroretinoic acid. These results show that the floor plate is a local source of a ZPA-like polarizing signal, possibly a retinoid, which may regulate the pattern of cell differentiation in the developing CNS.