Although development of the adult Drosophila compound eye is very well understood, little is known about development of photoreceptors (PRs) in the simple larval eye. We show here that the larval eye is composed of 12 PRs, four of which express blue-sensitive rhodopsin5 (rh5) while the other eight contain green-sensitive rh6. This is similar to the 30:70 ratio of adult blue and green R8 cells. However, the stochastic choice of adult color PRs and the bistable loop of the warts and melted tumor suppressor genes that unambiguously specify rh5 and rh6 in R8 PRs are not involved in specification of larval PRs. Instead, primary PR precursors signal via EGFR to surrounding tissue to develop as secondary precursors, which will become Rh6-expressing PRs. EGFR signaling is required for the survival of the Rh6 subtype. Primary precursors give rise to the Rh5 subtype. Furthermore, the combinatorial action of the transcription factors Spalt, Seven-up, and Orthodenticle specifies the two PR subtypes. Therefore, even though the larval PRs and adult R8 PRs express the same rhodopsins (rh5 and rh6), they use very distinct mechanisms for their specification.

Entry into mitosis is accompanied by profound changes in cortical actomyosin organization. Here, we delineate a pathway downstream of the RhoGEF Pbl/Ect2 that directs this process in a model epithelium. Our data suggest that the release of Pbl/Ect2 from the nucleus at mitotic entry drives Rho-dependent activation of Myosin-II and, in parallel, induces a switch from Arp2/3 to Diaphanous-mediated cortical actin nucleation that depends on Cdc42, aPKC, and Par6. At the same time, the mitotic relocalization of these apical protein complexes to more lateral cell surfaces enables Cdc42/aPKC/Par6 to take on a mitosis-specific function-aiding the assembly of a relatively isotropic metaphase cortex. Together, these data reveal how the repolarization and remodeling of the actomyosin cortex are coordinated upon entry into mitosis to provide cells with the isotropic and rigid form they need to undergo faithful chromosome segregation and division in a crowded tissue environment.

In Drosophila epithelial cells, specification and maintenance of the zonula adherens (za) is crucial to ensure epithelial tissue integrity. This depends on the intertwined function of Bazooka (Baz), Par6-DaPKC, and the Crumbs (Crb)-Stardust (Sdt)-PATJ complex. However, the detailed molecular basis for the interplay between these factors during this process is not fully understood.

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.

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.

The ability of epithelial cells to assemble into sheets relies on their zonula adherens (ZA), a circumferential belt of adherens junction (AJ) material, which can be remodeled during development to shape organs. Here, we show that during ZA remodeling in a model neuroepithelial cell, the Cdc42 effector P21-activated kinase 4 (Pak4/Mbt) regulates AJ morphogenesis and stability through β-catenin (β-cat/Arm) phosphorylation. We find that β-catenin phosphorylation by Mbt, and associated AJ morphogenesis, is needed for the retention of the apical determinant Par3/Bazooka at the remodeling ZA. Importantly, this retention mechanism functions together with Par1-dependent lateral exclusion of Par3/Bazooka to regulate apical membrane differentiation. Our results reveal an important functional link between Pak4, AJ material morphogenesis, and polarity remodeling during organogenesis downstream of Par3.

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.

Polarized epithelia develop distinct cell surface domains, with the apical membrane acquiring characteristic morphological features such as microvilli. Cell polarization is driven by polarity determinants including the evolutionarily conserved partitioning-defective (PAR) proteins that are separated into distinct cortical domains. PAR protein segregation is thought to be a consequence of asymmetric actomyosin contractions. The mechanism of activation of apically polarized actomyosin contractility is unknown. Here we show that the Cdc42 effector MRCK activates myosin-II at the apical pole to segregate aPKC-Par6 from junctional Par3, defining the apical domain. Apically polarized MRCK-activated actomyosin contractility is reinforced by cooperation with aPKC-Par6 downregulating antagonistic RhoA-driven junctional actomyosin contractility, and drives polarization of cytosolic brush border determinants and apical morphogenesis. MRCK-activated polarized actomyosin contractility is required for apical differentiation and morphogenesis in vertebrate epithelia and Drosophila photoreceptors. Our results identify an apical origin of actomyosin-driven morphogenesis that couples cytoskeletal reorganization to PAR polarity signalling.

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.

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.

Neuronal polarity in the developing cortex begins during the early stages of neural progenitor migration toward the cortical plate and culminates with the specification of the axon and dendrites. Here, we demonstrate that the Ran-dependent nucleocytoplasmic transport machinery is essential for the establishment of cortical neuron polarity. We found that Ran-binding protein 1 (RanBP1) regulates axon specification and dendritic arborization in cultured neurons in vitro and radial neural migration in vivo. During axonogenesis, RanBP1 regulates the cytoplasmic levels of the polarity protein LKB1/Par4, and this is dependent on the nuclear export machinery. Our results show that downstream of RanBP1, LKB1 function is mediated by the STK25-GM130 pathway, which promotes axonogenesis through Golgi regulation. Our results indicate that the nucleocytoplasmic transport machinery is a main regulator of neuron polarity, including radial migration, and that the regulated export of LKB1 through RanBP1 is a limiting step of axonogenesis.

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.

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.