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.     

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.     

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.     

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.     

Drosophila Crumbs is a positional cue in photoreceptor adherens junctions and rhabdomeres

Drosophila Crumbs (Crb) is required for apical-basal polarity and is an apical determinant in embryonic epithelia. Here, we describe properties of Crb that control the position and integrity of the photoreceptor adherens junction and photosensitive organ, or rhabdomere. In contrast to normal photoreceptor adherens junctions and rhabdomeres, which span the depth of the retina, adherens junctions and rhabdomeres of Crb-deficient photoreceptors initially accumulate at the top of the retina and fail to maintain their integrity as they stretch to the retinal floor. We show that Crb controls localization of the adherens junction through its intracellular domain containing a putative binding site for a protein 4.1 superfamily protein (FERM). Although loss of Crb or overexpression of the FERM binding domain causes mislocalization of adherens junctions, they do not result in a significant loss of photoreceptor polarity. Mutations in CRB1, a human homologue of crb, are associated with photoreceptor degeneration in retinitis pigmentosa 12 (RP12) and Leber congenital amaurosis (LCA). The intracellular domain of CRB1 behaves similarly to its Drosophila counterpart when overexpressed in the fly eye. Our studies may provide clues for mechanisms of photoreceptor degeneration in RP12 and LCA.     

Crumbs, the Drosophila homologue of human CRB1/RP12, is essential for photoreceptor morphogenesis

The apical transmembrane protein Crumbs is a central regulator of epithelial apical-basal polarity in Drosophila. Loss-of-function mutations in the human homologue of Crumbs, CRB1 (RP12), cause recessive retinal dystrophies, including retinitis pigmentosa. Here we show that Crumbs and CRB1 localize to corresponding subdomains of the photoreceptor apical plasma membrane: the stalk of the Drosophila photoreceptor and the inner segment of mammalian photoreceptors. These subdomains support the morphogenesis and orientation of the photosensitive membrane organelles: rhabdomeres and outer segments, respectively. Drosophila Crumbs is required to maintain zonula adherens integrity during the rapid apical membrane expansion that builds the rhabdomere. Crumbs also regulates stalk development by stabilizing the membrane-associated spectrin cytoskeleton, a function mechanistically distinct from its role in epithelial apical-basal polarity. We propose that Crumbs is a central component of a molecular scaffold that controls zonula adherens assembly and defines the stalk as an apical membrane subdomain. Defects in such scaffolds may contribute to human CRB1-related retinal dystrophies.     

Efflux-dependent auxin gradients establish the apical-basal axis of Arabidopsis

Axis formation occurs in plants, as in animals, during early embryogenesis. However, the underlying mechanism is not known. Here we show that the first manifestation of the apical-basal axis in plants, the asymmetric division of the zygote, produces a basal cell that transports and an apical cell that responds to the signalling molecule auxin. This apical-basal auxin activity gradient triggers the specification of apical embryo structures and is actively maintained by a novel component of auxin efflux, PIN7, which is located apically in the basal cell. Later, the developmentally regulated reversal of PIN7 and onset of PIN1 polar localization reorganize the auxin gradient for specification of the basal root pole. An analysis of pin quadruple mutants identifies PIN-dependent transport as an essential part of the mechanism for embryo axis formation. Our results indicate how the establishment of cell polarity, polar auxin efflux and local auxin response result in apical-basal axis formation of the embryo, and thus determine the axiality of the adult plant.     

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.     

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.     

Drosophila Stardust is a partner of Crumbs in the control of epithelial cell polarity.

The polarized architecture of epithelial cells depends on the highly stereotypic distribution of cellular junctions and other membrane-associated protein complexes. In epithelial cells of the Drosophila embryo, three distinct domains subdivide the lateral plasma membrane. The most apical one comprises the subapical complex (SAC). It is followed by the zonula adherens (ZA) and, further basally, by the septate junction. A core component of the SAC is the transmembrane protein Crumbs, the cytoplasmic domain of which recruits the PDZ-protein Discs Lost into the complex. Cells lacking crumbs or the functionally related gene stardust fail to organize a continuous ZA and to maintain cell polarity. Here we show that stardust provides an essential component of the SAC. Stardust proteins colocalize with Crumbs and bind to the carboxy-terminal amino acids of its cytoplasmic tail. We introduce two different Stardust proteins here: one MAGUK protein, characterized by a PDZ domain, an SH3 domain and a guanylate kinase domain; and a second isoform comprising only the guanylate kinase domain. The Stardust proteins represent versatile candidates as structural and possibly regulatory constituents of the SAC, a crucial element in the control of epithelial cell polarity.     

Moesin functions antagonistically to the Rho pathway to maintain epithelial integrity

Two prominent characteristics of epithelial cells, apical-basal polarity and a highly ordered cytoskeleton, depend on the existence of precisely localized protein complexes associated with the apical plasma membrane, and on a separate machinery that regulates the spatial order of actin assembly. ERM (ezrin, radixin, moesin) proteins have been proposed to link transmembrane proteins to the actin cytoskeleton in the apical domain, suggesting a structural role in epithelial cells, and they have been implicated in signalling pathways. Here, we show that the sole Drosophila ERM protein Moesin functions to promote cortical actin assembly and apical-basal polarity. As a result, cells lacking Moesin lose epithelial characteristics and adopt invasive migratory behaviour. Our data demonstrate that Moesin facilitates epithelial morphology not by providing an essential structural function, but rather by antagonizing activity of the small GTPase Rho. Thus, Moesin functions in maintaining epithelial integrity by regulating cell-signalling events that affect actin organization and polarity. Furthermore, our results show that there is negative feedback between ERM activation and activity of the Rho pathway.     

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.     

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.     

Centrosomes direct cell polarity independently of microtubule assembly in C. elegans embryos

Polarity establishment requires a symmetry-breaking event, resulting in an axis along which determinants are segregated. In Caenorhabditis elegans, oocytes are apolar and are triggered to polarize rapidly along one axis after fertilization. The establishment of this first polarity axis is revealed by the asymmetric distribution of PAR proteins and cortical activity in the one-celled embryo. Current evidence suggests that the centrosome-pronucleus complex contributed by the sperm is involved in defining the polarization axis. Here we directly assess the contribution of the centrosome to polarity establishment by laser ablating the centrosome before and during polarization. We find that the centrosome is required to initiate polarity but not to maintain it. Initiation of polarity coincides with the proximity of the centrosome to the cortex and the assembly of pericentriolar material on the immature sperm centrosome. Depletion of microtubules or the microtubule nucleator gamma-tubulin did not affect polarity establishment. These results demonstrate that the centrosome provides an initiating signal that polarizes C. elegans embryos and indicate that this signalling event might be independent of the role of the centrosome as a microtubule nucleator.     

Nodal signalling in the epiblast patterns the early mouse embryo.

Shortly after implantation the mouse embryo comprises three tissue layers. The founder tissue of the embryo proper, the epiblast, forms a radially symmetric cup of epithelial cells that grows in close apposition to the extra-embryonic ectoderm and the visceral endoderm. This simple cylindrical structure exhibits a distinct molecular pattern along its proximal-distal axis. The anterior-posterior axis of the embryo is positioned later by coordinated cell movements that rotate the pre-existing proximal-distal axis. The transforming growth factor-beta family member Nodal is known to be required for formation of the anterior-posterior axis. Here we show that signals from the epiblast are responsible for the initiation of proximal-distal polarity. Nodal acts to promote posterior cell fates in the epiblast and to maintain molecular pattern in the adjacent extra-embryonic ectoderm. Both of these functions are independent of Smad2. Moreover, Nodal signals from the epiblast also pattern the visceral endoderm by activating the Smad2-dependent pathway required for specification of anterior identity in overlying epiblast cells. Our experiments show that proximal-distal and subsequent anterior-posterior polarity of the pregastrulation embryo result from reciprocal cell-cell interactions between the epiblast and the two extra-embryonic tissues.     

A role for Drosophila LKB1 in anterior-posterior axis formation and epithelial polarity

The PAR-4 and PAR-1 kinases are necessary for the formation of the anterior-posterior (A-P) axis in Caenorhabditis elegans. PAR-1 is also required for A-P axis determination in Drosophila. Here we show that the Drosophila par-4 homologue, lkb1, is required for the early A-P polarity of the oocyte, and for the repolarization of the oocyte cytoskeleton that defines the embryonic A-P axis. LKB1 is phosphorylated by PAR-1 in vitro, and overexpression of LKB1 partially rescues the par-1 phenotype. These two kinases therefore function in a conserved pathway for axis formation in flies and worms. lkb1 mutant clones also disrupt apical-basal epithelial polarity, suggesting a general role in cell polarization. The human homologue, LKB1, is mutated in Peutz-Jeghers syndrome and is regulated by prenylation and by phosphorylation by protein kinase A. We show that protein kinase A phosphorylates Drosophila LKB1 on a conserved site that is important for its activity. Thus, Drosophila and human LKB1 may be functional homologues, suggesting that loss of cell polarity may contribute to tumour formation in individuals with Peutz-Jeghers syndrome.     

Drosophila Stardust interacts with Crumbs to control polarity of epithelia but not neuroblasts.

Establishing cellular polarity is critical for tissue organization and function. Initially discovered in the landmark genetic screen for Drosophila developmental mutants, bazooka, crumbs, shotgun and stardust mutants exhibit severe disruption in apicobasal polarity in embryonic epithelia, resulting in multilayered epithelia, tissue disintegration, and defects in cuticle formation. Here we report that stardust encodes single PDZ domain MAGUK (membrane-associated guanylate kinase) proteins that are expressed in all primary embryonic epithelia from the onset of gastrulation. Stardust colocalizes with Crumbs at the apicolateral boundary, although their expression patterns in sensory organs differ. Stardust binds to the carboxy terminus of Crumbs in vitro, and Stardust and Crumbs are mutually dependent in their stability, localization and function in controlling the apicobasal polarity of epithelial cells. However, for the subset of ectodermal cells that delaminate and form neuroblasts, their polarity requires the function of Bazooka, but not of Stardust or Crumbs.     

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.     

A positive feedback mechanism governs the polarity and motion of motile cilia

Ciliated epithelia produce fluid flow in many organ systems, ranging from the respiratory tract where it clears mucus to the ventricles of the brain where it transports cerebrospinal fluid. Human diseases that disable ciliary flow, such as primary ciliary dyskinesia, can compromise organ function or the ability to resist pathogens, resulting in recurring respiratory infections, otitis, hydrocephaly and infertility. To create a ciliary flow, the cilia within each cell need to be polarized coordinately along the planar axis of the epithelium, but how polarity is established in any ciliated epithelia is not known. Here we analyse the developmental mechanisms that polarize cilia, using the ciliated cells in the developing Xenopus larval skin as a model system. We show that cilia acquire polarity through a sequence of events, beginning with a polar bias set by tissue patterning, followed by a refinement phase. Our results indicate that during refinement, fluid flow is both necessary and sufficient in determining cilia polarity. These findings reveal a novel mechanism in which tissue patterning coupled with fluid flow act in a positive feedback loop to direct the planar polarity of cilia.