Sticking together the Crumbs - an unexpected function for an old friend.

Cell polarity and cell-cell junctions have pivotal roles in organizing cells into tissues and in mediating cell-cell communication. The transmembrane protein Crumbs has a well-established role in the maintenance of epithelial polarity, and it can also regulate signalling via the Notch and Hippo pathways to influence tissue growth. The functions of Crumbs in epithelial polarity and Hippo-mediated growth depend on its short intracellular domain. Recent evidence now points to a conserved and fundamental role for the extracellular domain of Crumbs in mediating homophilic Crumbs-Crumbs interactions at cell-cell junctions.

Shaping an optical dome: The size and shape of the insect compound eye.

The insect compound eye is the most abundant eye architecture on earth. It comes in a wide variety of shapes and sizes, which are exquisitely adapted to specific ecosystems. Here, we explore the organisational principles and pathways, from molecular to tissular, that underpin the building of this organ and highlight why it is an excellent model system to investigate the relationship between genes and tissue form. The compound eye offers wide fields of view, high sensitivity in motion detection and infinite depth of field. It is made of an array of visual units called ommatidia, which are precisely tiled in 3D to shape the retinal tissue as a dome-like structure. The eye starts off as a 2D epithelium, and it acquires its 3D organisation as ommatidia get into shape. Each ommatidium is made of a complement of retinal cells, including light-detecting photoreceptors and lens-secreting cells. The lens cells generate the typical hexagonal facet lens that lies atop the photoreceptors so that the eye surface consists of a quasi-crystalline array of these hexagonal facet-lenses. This array is curved to various degree, depending on the size and shape of the eye, and on the region of the retina. This curvature sets the resolution and visual field of the eye and is determined by i) the number and size of the facet lens - large ommatidial lenses can be used to generate flat, higher resolution areas, while smaller facets allow for stronger curvature of the eye, and ii) precise control of the inter facet-lens angle, which determines the optical axis of the each ommatidium. In this review we discuss how combinatorial variation in eye primordium shape, ommatidial number, facet lens size and inter facet-lens angle underpins the wide variety of insect eye shapes, and we explore what is known about the mechanisms that might control these parameters.

A combination of Notch signaling, preferential adhesion and endocytosis induces a slow mode of cell intercalation in the Drosophila retina.

Movement of epithelial cells in a tissue occurs through neighbor exchange and drives tissue shape changes. It requires intercellular junction remodeling, a process typically powered by the contractile actomyosin cytoskeleton. This has been investigated mainly in homogeneous epithelia, where intercalation takes minutes. However, in some tissues, intercalation involves different cell types and can take hours. Whether slow and fast intercalation share the same mechanisms remains to be examined. To address this issue, we used the fly eye, where the cone cells exchange neighbors over ∼10 h to shape the lens. We uncovered three pathways regulating this slow mode of cell intercalation. First, we found a limited requirement for MyosinII. In this case, mathematical modeling predicts an adhesion-dominant intercalation mechanism. Genetic experiments support this prediction, revealing a role for adhesion through the Nephrin proteins Roughest and Hibris. Second, we found that cone cell intercalation is regulated by the Notch pathway. Third, we show that endocytosis is required for membrane removal and Notch activation. Taken together, our work indicates that adhesion, endocytosis and Notch can direct slow cell intercalation during tissue morphogenesis.

RanBP1 plays an essential role in directed migration of neural crest cells during development.

Collective cell migration is essential for embryonic development, tissue regeneration and repair, and has been implicated in pathological conditions such as cancer metastasis. It is, in part, directed by external cues that promote front-to-rear polarity in individual cells. However, our understanding of the pathways that underpin the directional movement of cells in response to external cues remains incomplete. To examine this issue we made use of neural crest cells (NC), which migrate as a collective during development to generate vital structures including bones and cartilage. Using a candidate approach, we found an essential role for Ran-binding protein 1 (RanBP1), a key effector of the nucleocytoplasmic transport pathway, in enabling directed migration of these cells. Our results indicate that RanBP1 is required for establishing front-to-rear polarity, so that NCs are able to chemotax. Moreover, our work suggests that RanBP1 function in chemotaxis involves the polarity kinase LKB1/PAR4. We envisage that regulated nuclear export of LKB1 through Ran/RanBP1 is a key regulatory step required for establishing front-to-rear polarity and thus chemotaxis, during NC collective migration.

Cdc42 defines apical identity and regulates epithelial morphogenesis by promoting apical recruitment of Par6-aPKC and Crumbs.

Cdc42 regulates epithelial morphogenesis together with the Par complex (Baz/Par3-Par6-aPKC), Crumbs (Crb/CRB3) and Stardust (Sdt/PALS1). However, how these proteins work together and interact during epithelial morphogenesis is not well understood. To address this issue, we used the genetically amenable Drosophila pupal photoreceptor and follicular epithelium. We show that during epithelial morphogenesis active Cdc42 accumulates at the developing apical membrane and cell-cell contacts, independently of the Par complex and Crb. However, membrane localization of Baz, Par6-aPKC and Crb all depend on Cdc42. We find that although binding of Cdc42 to Par6 is not essential for the recruitment of Par6 and aPKC to the membrane, it is required for their apical localization and accumulation, which we find also depends on Par6 retention by Crb. In the pupal photoreceptor, membrane recruitment of Par6-aPKC also depends on Baz. Our work shows that Cdc42 is required for this recruitment and suggests that this factor promotes the handover of Par6-aPKC from Baz onto Crb. Altogether, we propose that Cdc42 drives morphogenesis by conferring apical identity, Par-complex assembly and apical accumulation of Crb.

Regulation of Cdc42 and its effectors in epithelial morphogenesis.

Cdc42 - a member of the small Rho GTPase family - regulates cell polarity across organisms from yeast to humans. It is an essential regulator of polarized morphogenesis in epithelial cells, through coordination of apical membrane morphogenesis, lumen formation and junction maturation. In parallel, work in yeast and Caenorhabditiselegans has provided important clues as to how this molecular switch can generate and regulate polarity through localized activation or inhibition, and cytoskeleton regulation. Recent studies have revealed how important and complex these regulations can be during epithelial morphogenesis. This complexity is mirrored by the fact that Cdc42 can exert its function through many effector proteins. In epithelial cells, these include atypical PKC (aPKC, also known as PKC-3), the P21-activated kinase (PAK) family, myotonic dystrophy-related Cdc42 binding kinase beta (MRCKß, also known as CDC42BPB) and neural Wiskott-Aldrich syndrome protein (N-WASp, also known as WASL). Here, we review how the spatial regulation of Cdc42 promotes polarity and polarized morphogenesis of the plasma membrane, with a focus on the epithelial cell type.

Rap1, Canoe and Mbt cooperate with Bazooka to promote zonula adherens assembly in the fly photoreceptor.

In Drosophila epithelial cells, apical exclusion of Bazooka (the Drosophila Par3 protein) defines the position of the zonula adherens (ZA), which demarcates the apical and lateral membrane and allows cells to assemble into sheets. Here, we show that the small GTPase Rap1, its effector Canoe (Cno) and the Cdc42 effector kinase Mushroom bodies tiny (Mbt), converge in regulating epithelial morphogenesis by coupling stabilization of the adherens junction (AJ) protein E-Cadherin and Bazooka retention at the ZA. Furthermore, our results show that the localization of Rap1, Cno and Mbt at the ZA is interdependent, indicating that their functions during ZA morphogenesis are interlinked. In this context, we find the Rap1-GEF Dizzy is enriched at the ZA and our results suggest that it promotes Rap1 activity during ZA morphogenesis. Altogether, we propose the Dizzy, Rap1 and Cno pathway and Mbt converge in regulating the interface between Bazooka and AJ material to promote ZA morphogenesis.

Orthodenticle Is Required for the Expression of Principal Recognition Molecules That Control Axon Targeting in the Drosophila Retina.

Parallel processing of neuronal inputs relies on assembling neural circuits into distinct synaptic-columns and layers. This is orchestrated by matching recognition molecules between afferent growth cones and target areas. Controlling the expression of these molecules during development is crucial but not well understood. The developing Drosophila visual system is a powerful genetic model for addressing this question. In this model system, the achromatic R1-6 photoreceptors project their axons in the lamina while the R7 and R8 photoreceptors, which are involved in colour detection, project their axons to two distinct synaptic-layers in the medulla. Here we show that the conserved homeodomain transcription factor Orthodenticle (Otd), which in the eye is a main regulator of rhodopsin expression, is also required for R1-6 photoreceptor synaptic-column specific innervation of the lamina. Our data indicate that otd function in these photoreceptors is largely mediated by the recognition molecules flamingo (fmi) and golden goal (gogo). In addition, we find that otd regulates synaptic-layer targeting of R8. We demonstrate that during this process, otd and the R8-specific transcription factor senseless/Gfi1 (sens) function as independent transcriptional inputs that are required for the expression of fmi, gogo and the adhesion molecule capricious (caps), which govern R8 synaptic-layer targeting. Our work therefore demonstrates that otd is a main component of the gene regulatory network that regulates synaptic-column and layer targeting in the fly visual system.

Transcriptional regulation of tissue organization and cell morphogenesis: the fly retina as a case study.

Understanding how a functional organ can be produced from a small group of cells remains an outstanding question in cell and developmental biology. The developing compound eye of Drosophila has long been a model of choice for addressing this question by dissecting the cellular, genetic and molecular pathways that govern cell specification, differentiation, and multicellular patterning during organogenesis. In this review, the author focussed on cell and tissue morphogenesis during fly retinal development, including the regulated changes in cell shape and cell packing that ultimately determine the shape and architecture of the compound eye. In particular, the author reviewed recent studies that highlight the prominent roles of transcriptional and hormonal controls that orchestrate the cell shape changes, cell-cell junction remodeling and polarized membrane growth that underlie photoreceptor morphogenesis and retinal patterning.

Atonal and EGFR signalling orchestrate rok- and Drak-dependent adherens junction remodelling during ommatidia morphogenesis.

Morphogenesis of epithelial tissues relies on the interplay between cell division, differentiation and regulated changes in cell shape, intercalation and sorting. These processes are often studied individually in relatively simple epithelia that lack the complexity found during organogenesis when these processes might all coexist simultaneously. To address this issue, we are making use of the developing fly retinal neuroepithelium. Retinal morphogenesis relies on a coordinated sequence of interdependent morphogenetic events that includes apical cell constriction, localized alignment of groups of cells and ommatidia morphogenesis coupled to neurogenesis. Here, we use live imaging to document the sequence of adherens junction (AJ) remodelling events required to generate the fly ommatidium. In this context, we demonstrate that the kinases Rok and Drak function redundantly during Myosin II-dependent cell constriction, subsequent multicellular alignment and AJ remodelling. In addition, we show that early multicellular patterning characterized by cell alignment is promoted by the conserved transcription factor Atonal (Ato). Further ommatidium patterning requires the epidermal growth factor receptor (EGFR) signalling pathway, which transcriptionally governs rok- and Drak-dependent AJ remodelling while also promoting neurogenesis. In conclusion, our work reveals an important role for Drak in regulating AJ remodelling during retinal morphogenesis. It also sheds new light on the interplay between Ato, EGFR-dependent transcription and AJ remodelling in a system in which neurogenesis is coupled with cell shape changes and regulated steps of cell intercalation.

Orthodenticle and Kruppel homolog 1 regulate Drosophila photoreceptor maturation.

Neurons present a wide variety of morphologies that are associated with their specialized functions. However, to date very few pathways and factors regulating neuronal maturation, including morphogenesis, have been identified. To address this issue we make use here of the genetically amenable developing fly photoreceptor (PR). Whereas this sensory neuron is specified early during retinal development, its maturation spans several days. During this time, this neuron acquires specialized membrane domains while undergoing extensive polarity remodeling. In this study, we identify a pathway in which the conserved homeobox protein Orthodenticle (Otd) acts together with the ecdysone receptor (EcR) to directly repress the expression of the transcription factor (TF) Kruppel homolog 1 (Kr-h1). We demonstrate that this pathway is not required to promote neuronal specification but is crucial to regulate PR maturation. PR maturation includes the remodeling of the cell's epithelial features and associated apical membrane morphogenesis. Furthermore, we show that hormonal control coordinates PR differentiation and morphogenesis with overall development. This study demonstrates that during PR differentiation, transient repression of Kr-h1 represents a key step regulating neuronal maturation. Down-regulation of Kr-h1 expression has been previously associated with instances of neuronal remodeling in the fly brain. We therefore conclude that repression of this transcription factor represents a key step, enabling remodeling and maturation in a wide variety of neurons.

Dynamin- and Rab5-dependent endocytosis is required to prevent Drosophila photoreceptor degeneration.

In Drosophila photoreceptors, Rhodopsin 1 (ninaE, Rh1) is required for proper morphogenesis and maintenance of the apical light-gathering organelle, the rhabdomere. It has been proposed that Rh1, coupled to the Rho GTPases Rac1 and Cdc42, promotes the morphogenesis of a sub-rhabdomeric F-actin meshwork or rhabdomere terminal web (RTW). The RTW provides mechanical support to the apical microvilli and is likely to guide Rab11-dependent delivery of Rh1-rich membrane to the rhabdomere from the trans Golgi network. However, the nature and function of the molecular pathway involved in RTW morphogenesis remains incomplete. Here, we show that Rh1 function in promoting RTW morphogenesis is light-independent and is conserved throughout evolution. This Rh1 function does not require G(q)α(e), which is required for phototransduction. Finally, we show that interfering with Dynamin- and Rab5-dependent endocytosis leads to a phenotype that is undistinguishable from that of the ninaE-null mutant. Importantly, the corresponding endocytic activity is essential to prevent early onset of rhabdomere degeneration. In conclusion, we propose that Rh1 function in promoting RTW morphogenesis is not only needed to sustain apical membrane delivery but is also required for proper rhabdomeric membrane endocytosis and turnover.

Crumbs is required to achieve proper organ size control during Drosophila head development.

Crumbs (Crb) is a conserved apical polarity determinant required for zonula adherens specification and remodelling during Drosophila development. Interestingly, crb function in maintaining apicobasal polarity appears largely dispensable in primary epithelia such as the imaginal discs. Here, we show that crb function is not required for maintaining epithelial integrity during the morphogenesis of the Drosophila head and eye. However, although crb mutant heads are properly developed, they are also significantly larger than their wild-type counterparts. We demonstrate that in the eye, this is caused by an increase in cell proliferation that can be attributed to an increase in ligand-dependent Notch (N) signalling. Moreover, we show that in crb mutant cells, ectopic N activity correlates with an increase in N and Delta endocytosis. These data indicate a role for Crb in modulating endocytosis at the apical epithelial plasma membrane, which we demonstrate is independent of Crb function in apicobasal polarity. Overall, our work reveals a novel function for Crb in limiting ligand-dependent transactivation of the N receptor at the epithelial cell membrane.

Hedgehog signaling is a principal inducer of Myosin-II-driven cell ingression in Drosophila epithelia.

Cell constriction promotes epithelial sheet invagination during embryogenesis across phyla. However, how this cell response is linked to global patterning information during organogenesis remains unclear. To address this issue, we have used the Drosophila eye and studied the formation of the morphogenetic furrow (MF), which is characterized by cells undergoing a synchronous apical constriction and apicobasal contraction. We show that this cell response relies on microtubules and F-actin enrichment within the apical domain of the constricting cell as well as on the activation of nonmuscle myosin. In the MF, Hedgehog (Hh) signaling is required to promote cell constriction downstream of cubitus interruptus (ci), and, in this context, Ci155 functions redundantly with mad, the main effector of dpp/BMP signaling. Furthermore, ectopically activating Hh signaling in fly epithelia reveals a direct relationship between the duration of exposure to this signaling pathway, the accumulation of activated Myosin II, and the degree of tissue invagination.

p32 is a novel mammalian Lgl binding protein that enhances the activity of protein kinase Czeta and regulates cell polarity.

Lgl (lethal giant larvae) plays an important role in cell polarity. Atypical protein kinase C (aPKC) binds to and phosphorylates Lgl, and the phosphorylation negatively regulates Lgl activity. In this study, we identify p32 as a novel Lgl binding protein that directly binds to a domain on mammalian Lgl2 (mLgl2), which contains the aPKC phosphorylation site. p32 also binds to PKCzeta, and the three proteins form a transient ternary complex. When p32 is bound, PKCzeta is stimulated to phosphorylate mLgl2 more efficiently. p32 overexpression in Madin-Darby canine kidney cells cultured in a 3D matrix induces an expansion of the actin-enriched apical membrane domain and disrupts cell polarity. Addition of PKCzeta inhibitor blocks apical actin accumulation, which is rescued by p32 overexpression. p32 knockdown by short hairpin RNA also induces cell polarity defects. Collectively, our data indicate that p32 is a novel regulator of cell polarity that forms a complex with mLgl2 and aPKC and enhances aPKC activity.

The ultrastructural organization of endoplasmic reticulum-plasma membrane contacts is conserved in epithelial cells.

Contacts between the endoplasmic reticulum and the plasma membrane (ER-PM contacts) have important roles in membrane lipid and calcium dynamics, yet their organization in polarized epithelial cells has not been thoroughly described. Here we examine ER-PM contacts in hepatocytes in mouse liver using electron microscopy, providing the first comprehensive ultrastructural study of ER-PM contacts in a mammalian epithelial tissue. Our quantitative analyses reveal strikingly distinct ER-PM contact architectures spatially linked to apical, lateral, and basal PM domains. Notably, we find that an extensive network of ER-PM contacts exists at lateral PM domains that form intercellular junctions between hepatocytes. Moreover, the spatial organization of ER-PM contacts is conserved in epithelial spheroids, suggesting that ER-PM contacts may serve conserved roles in epithelial cell architecture. Consistent with this notion, we show that ORP5 activity at ER-PM contacts modulates the apical-basolateral aspect ratio in HepG2 cells. Thus ER-PM contacts have a conserved distribution and crucial roles in PM domain architecture across epithelial cell types.

Iroquois complex genes induce co-expression of rhodopsins in Drosophila.

The Drosophila eye is a mosaic that results from the stochastic distribution of two ommatidial subtypes. Pale and yellow ommatidia can be distinguished by the expression of distinct rhodopsins and other pigments in their inner photoreceptors (R7 and R8), which are implicated in color vision. The pale subtype contains ultraviolet (UV)-absorbing Rh3 in R7 and blue-absorbing Rh5 in R8. The yellow subtype contains UV-absorbing Rh4 in R7 and green-absorbing Rh6 in R8. The exclusive expression of one rhodopsin per photoreceptor is a widespread phenomenon, although exceptions exist. The mechanisms leading to the exclusive expression or to co-expression of sensory receptors are currently not known. We describe a new class of ommatidia that co-express rh3 and rh4 in R7, but maintain normal exclusion between rh5 and rh6 in R8. These ommatidia, which are localized in the dorsal eye, result from the expansion of rh3 into the yellow-R7 subtype. Genes from the Iroquois Complex (Iro-C) are necessary and sufficient to induce co-expression in yR7. Iro-C genes allow photoreceptors to break the "one receptor-one neuron" rule, leading to a novel subtype of broad-spectrum UV- and green-sensitive ommatidia.

Sec15 interacts with Rab11 via a novel domain and affects Rab11 localization in vivo.

Sec15, a component of the exocyst, recognizes vesicle-associated Rab GTPases, helps target transport vesicles to the budding sites in yeast and is thought to recruit other exocyst proteins. Here we report the characterization of a 35-kDa fragment that comprises most of the C-terminal half of Drosophila melanogaster Sec15. This C-terminal domain was found to bind a subset of Rab GTPases, especially Rab11, in a GTP-dependent manner. We also provide evidence that in fly photoreceptors Sec15 colocalizes with Rab11 and that loss of Sec15 affects rhabdomere morphology. Determination of the 2.5-A crystal structure of the C-terminal domain revealed a novel fold consisting of ten alpha-helices equally distributed between two subdomains (N and C subdomains). We show that the C subdomain, mainly via a single helix, is sufficient for Rab binding.

Cell-type-specific mechanical response and myosin dynamics during retinal lens development in Drosophila.

During organogenesis, different cell types need to work together to generate functional multicellular structures. To study this process, we made use of the genetically tractable fly retina, with a focus on the mechanisms that coordinate morphogenesis between the different epithelial cell types that make up the optical lens. Our work shows that these epithelial cells present contractile apical-medial MyosinII meshworks, which control the apical area and junctional geometry of these cells during lens development. Our study also suggests that these MyosinII meshworks drive cell shape changes in response to external forces, and thus they mediate part of the biomechanical coupling that takes place between these cells. Importantly, our work, including mathematical modeling of forces and material stiffness during lens development, raises the possibility that increased cell stiffness acts as a mechanism for limiting this mechanical coupling. We propose this might be required in complex tissues, where different cell types undergo concurrent morphogenesis and where averaging out of forces across cells could compromise individual cell apical geometry and thereby organ function.