Confinement promotes nematic alignment of spindle-shaped cells during Drosophila embryogenesis.

Tissue morphogenesis is often controlled by actomyosin networks pulling on adherens junctions (AJs), but junctional myosin levels vary. At an extreme, the Drosophila embryo amnioserosa forms a horseshoe-shaped strip of aligned, spindle-shaped cells lacking junctional myosin. What are the bases of amnioserosal cell interactions and alignment? Compared with surrounding tissue, we find that amnioserosal AJ continuity has lesser dependence on α-catenin, the mediator of AJ-actomyosin association, and greater dependence on Bazooka/Par-3, a junction-associated scaffold protein. Microtubule bundles also run along amnioserosal AJs and support their long-range curvilinearity. Amnioserosal confinement is apparent from partial overlap of its spindle-shaped cells, its outward bulging from surrounding tissue and from compressive stress detected within the amnioserosa. Genetic manipulations that alter amnioserosal confinement by surrounding tissue also result in amnioserosal cells losing alignment and gaining topological defects characteristic of nematically ordered systems. With Bazooka depletion, confinement by surrounding tissue appears to be relatively normal and amnioserosal cells align despite their AJ fragmentation. Overall, the fully elongated amnioserosa appears to form through tissue-autonomous generation of spindle-shaped cells that nematically align in response to confinement by surrounding tissue.

Adherens junctions: from molecules to morphogenesis.

How adhesive interactions between cells generate and maintain animal tissue structure remains one of the most challenging and long-standing questions in cell and developmental biology. Adherens junctions (AJs) and the cadherin-catenin complexes at their core are therefore the subjects of intense research. Recent work has greatly advanced our understanding of the molecular organization of AJs and how cadherin-catenin complexes engage actin, microtubules and the endocytic machinery. As a result, we have gained important insights into the molecular mechanisms of tissue morphogenesis.

Sculpting epithelia with planar polarized actomyosin networks: Principles from Drosophila.

Drosophila research has revealed how planar polarized actomyosin networks affect various types of tissue morphogenesis. The networks are positioned by both tissue-wide patterning factors (including Even-skipped, Runt, Engrailed, Invected, Hedgehog, Notch, Wingless, Epidermal Growth Factor, Jun N-terminal kinase, Sex combs reduced and Fork head) and local receptor complexes (including Echinoid, Crumbs and Toll receptors). Networks with differing super-structure and contractile output have been discovered. Their contractility can affect individual cells or can be coordinated across groups of cells, and such contractility can drive or resist physical change. For what seem to be simple tissue changes, multiple types of actomyosin networks can contribute, acting together as contractile elements or braces within the developing structure. This review discusses the positioning and effects of planar polarized actomyosin networks for a number of developmental events in Drosophila, including germband extension, dorsal closure, head involution, tracheal pit formation, salivary gland development, imaginal disc boundary formation, and tissue rotation of the male genitalia and the egg chamber.

Dynamics of PAR Proteins Explain the Oscillation and Ratcheting Mechanisms in Dorsal Closure.

We present a vertex-based model for Drosophila dorsal closure that predicts the mechanics of cell oscillation and contraction from the dynamics of the PAR proteins. Based on experimental observations of how aPKC, Par-6, and Bazooka translocate from the circumference of the apical surface to the medial domain, and how they interact with each other and ultimately regulate the apicomedial actomyosin, we formulate a system of differential equations that captures the key features of dorsal closure, including distinctive behaviors in its early, slow, and fast phases. The oscillation in cell area in the early phase of dorsal closure results from an intracellular negative feedback loop that involves myosin, an actomyosin regulator, aPKC, and Bazooka. In the slow phase, gradual sequestration of apicomedial aPKC by Bazooka clusters causes incomplete disassembly of the actomyosin network over each cycle of oscillation, thus producing a so-called ratchet. The fast phase of rapid cell and tissue contraction arises when medial myosin, no longer antagonized by aPKC, builds up in time and produces sustained contraction. Thus, a minimal set of rules governing the dynamics of the PAR proteins, extracted from experimental observations, can account for all major mechanical outcomes of dorsal closure, including the transitions between its three distinct phases.

Cadherin Trafficking for Tissue Morphogenesis: Control and Consequences.

Cadherin-based adherens junctions are critical for connecting cells in tissues. Regulated cadherin trafficking also makes these complexes amazingly dynamic, with permissive and instructive consequences on multicellular development. Here, we review how cadherin trafficking affects various forms of tissue morphogenesis from Drosophila and Caenorhabditis elegans to zebrafish, Xenopus and mouse. We describe how core trafficking machinery (such as clathrin, dynamin, Rab small G proteins and the exocyst complex) integrates with other molecular systems (transcriptional factors, signaling pathways, microtubules, actin networks, apico-basal polarity proteins and planar cell polarity proteins) to control cadherin endocytosis, exocytosis and recycling. This control can occur at all cell-cell contacts or specific junctions for distinct effects on tissue morphogenesis during animal development.

Bazooka inhibits aPKC to limit antagonism of actomyosin networks during amnioserosa apical constriction.

Cell shape changes drive tissue morphogenesis during animal development. An important example is the apical cell constriction that initiates tissue internalisation. Apical constriction can occur through a phase of cyclic assembly and disassembly of apicomedial actomyosin networks, followed by stabilisation of these networks. Delayed negative-feedback mechanisms typically underlie cyclic behaviour, but the mechanisms regulating cyclic actomyosin networks remain obscure, as do mechanisms that transform overall network behaviour. Here, we show that a known inhibitor of apicomedial actomyosin networks in Drosophila amnioserosa cells, the Par-6-aPKC complex, is recruited to the apicomedial domain by actomyosin networks during dorsal closure of the embryo. This finding establishes an actomyosin-aPKC negative-feedback loop in the system. Additionally, we find that aPKC recruits Bazooka to the apicomedial domain, and phosphorylates Bazooka for a dynamic interaction. Remarkably, stabilising aPKC-Bazooka interactions can inhibit the antagonism of actomyosin by aPKC, suggesting that Bazooka acts as an aPKC inhibitor, and providing a possible mechanism for delaying the actomyosin-aPKC negative-feedback loop. Our data also implicate an increasing degree of Par-6-aPKC-Bazooka interactions as dorsal closure progresses, potentially explaining a developmental transition in actomyosin behaviour from cyclic to persistent networks. This later impact of aPKC inhibition is supported by mathematical modelling of the system. Overall, this work illustrates how shifting chemical signals can tune actomyosin network behaviour during development.

Assembly of Bazooka polarity landmarks through a multifaceted membrane-association mechanism.

Epithelial cell polarity is essential for animal development. The scaffold protein Bazooka (Baz/PAR-3) forms apical polarity landmarks to organize epithelial cells. However, it is unclear how Baz is recruited to the plasma membrane and how this is coupled with downstream effects. Baz contains an oligomerization domain, three PDZ domains, and binding regions for the protein kinase aPKC and phosphoinositide lipids. With a structure-function approach, we dissected the roles of these domains in the localization and function of Baz in the Drosophila embryonic ectoderm. We found that a multifaceted membrane association mechanism localizes Baz to the apical circumference. Although none of the Baz protein domains are essential for cortical localization, we determined that each contributes to cortical anchorage in a specific manner. We propose that the redundancies involved might provide plasticity and robustness to Baz polarity landmarks. We also identified specific downstream effects, including the promotion of epithelial structure, a positive-feedback loop that recruits aPKC, PAR-6 and Crumbs, and a negative-feedback loop that regulates Baz.

Centrosome-organized plasma membrane infoldings linked to growth of a cortical actin domain.

Regulated cell shape change requires the induction of cortical cytoskeletal domains. Often, local changes to plasma membrane (PM) topography are involved. Centrosomes organize cortical domains and can affect PM topography by locally pulling the PM inward. Are these centrosome effects coupled? At the syncytial Drosophila embryo cortex, centrosome-induced actin caps grow into dome-like compartments for mitoses. We found the nascent cap to be a collection of PM folds and tubules formed over the astral centrosomal MT array. The localized infoldings require centrosome and dynein activities, and myosin-based surface tension prevents them elsewhere. Centrosome-engaged PM infoldings become specifically enriched with an Arp2/3 induction pathway. Arp2/3 actin network growth between the infoldings counterbalances centrosomal pulling forces and disperses the folds for actin cap expansion. Abnormal domain topography with either centrosome or Arp2/3 disruption correlates with decreased exocytic vesicle association. Together, our data implicate centrosome-organized PM infoldings in coordinating Arp2/3 network growth and exocytosis for cortical domain assembly.

The PAR complex regulates pulsed actomyosin contractions during amnioserosa apical constriction in Drosophila.

Apical constriction is a major mechanism underlying tissue internalization during development. This cell constriction typically requires actomyosin contractility. Thus, understanding apical constriction requires characterization of the mechanics and regulation of actomyosin assemblies. We have analyzed the relationship between myosin and the polarity regulators Par-6, aPKC and Bazooka (Par-3) (the PAR complex) during amnioserosa apical constriction at Drosophila dorsal closure. The PAR complex and myosin accumulate at the apical surface domain of amnioserosa cells at dorsal closure, the PAR complex forming a patch of puncta and myosin forming an associated network. Genetic interactions indicate that the PAR complex supports myosin activity during dorsal closure, as well as during other steps of embryogenesis. We find that actomyosin contractility in amnioserosa cells is based on the repeated assembly and disassembly of apical actomyosin networks, with each assembly event driving constriction of the apical domain. As the networks assemble they translocate across the apical patch of PAR proteins, which persist at the apical domain. Through loss- and gain-of-function studies, we find that different PAR complex components regulate distinct phases of the actomyosin assembly/disassembly cycle: Bazooka promotes the duration of actomyosin pulses and Par-6/aPKC promotes the lull time between pulses. These results identify the mechanics of actomyosin contractility that drive amnioserosa apical constriction and how specific steps of the contractile mechanism are regulated by the PAR complex.

An introduction to adherens junctions: from molecular mechanisms to tissue development and disease.

Adherens junctions (AJs) are fundamental for the development of animal tissues and organs. The core complex is formed from transmembrane cell-cell adhesion molecules, cadherins, and adaptor molecules, the catenins, that link to cytoskeletal and regulatory networks within the cell. This complex can be considered over a wide range of biological organization, from atoms to molecules, protein complexes, molecular networks, cells, tissues, and overall animal development. AJs have also been an integral part of animal evolution, and play central roles in cancer development and pathogen infection. This book addresses major questions encompassing these aspects of AJ biology. How did AJs evolve? How do the cadherins and catenins interact to assemble AJs and mediate adhesion? How do AJs interface with other cellular machinery to couple adhesion with the whole cell? How do AJs affect cell behaviour and multicellular development? How can abnormal AJ activity lead to disease?

Contractile and expansive actin networks in Drosophila: Developmental cell biology controlled by network polarization and higher-order interactions.

Actin networks are central to shaping and moving cells during animal development. Various spatial cues activate conserved signal transduction pathways to polarize actin network assembly at sub-cellular locations and to elicit specific physical changes. Actomyosin networks contract and Arp2/3 networks expand, and to affect whole cells and tissues they do so within higher-order systems. At the scale of tissues, actomyosin networks of epithelial cells can be coupled via adherens junctions to form supracellular networks. Arp2/3 networks typically integrate with distinct actin assemblies, forming expansive composites which act in conjunction with contractile actomyosin networks for whole-cell effects. This review explores these concepts using examples from Drosophila development. First, we discuss the polarized assembly of supracellular actomyosin cables which constrict and reshape epithelial tissues during embryonic wound healing, germ band extension, and mesoderm invagination, but which also form physical borders between tissue compartments at parasegment boundaries and during dorsal closure. Second, we review how locally induced Arp2/3 networks act in opposition to actomyosin structures during myoblast cell-cell fusion and cortical compartmentalization of the syncytial embryo, and how Arp2/3 and actomyosin networks also cooperate for the single cell migration of hemocytes and the collective migration of border cells. Overall, these examples show how the polarized deployment and higher-order interactions of actin networks organize developmental cell biology.

aPKC controls microtubule organization to balance adherens junction symmetry and planar polarity during development.

Tissue morphogenesis requires assembling and disassembling individual cell-cell contacts without losing epithelial integrity. This requires dynamic control of adherens junction (AJ) positioning around the apical domain, but the mechanisms involved are unclear. We show that atypical Protein Kinase C (aPKC) is required for symmetric AJ positioning during Drosophila embryogenesis. aPKC is dispensable for initial apical AJ recruitment, but without aPKC, AJs form atypical planar-polarized puncta at gastrulation. Preceding this, microtubules fail to dissociate from centrosomes, and at gastrulation abnormally persistent centrosomal microtubule asters cluster AJs into the puncta. Dynein enrichment at the puncta suggests it may draw AJs and microtubules together and microtubule disruption disperses the puncta. Through cytoskeletal disruption in wild-type embryos, we find a balance of microtubule and actin interactions controls AJ symmetry versus planar polarity during normal gastrulation. aPKC apparently regulates this balance. Without aPKC, abnormally strong microtubule interactions break AJ symmetry and epithelial structure is lost.

Axis specification: Breaking symmetry with a myosin patch in the egg.

Drosophila anterior-posterior axis specification occurs in the oocyte, but the initial symmetry break has been unclear. A new study reveals that a posterior domain of cortical myosin is induced with unique post-translational modification and dynamics and that this domain recruits downstream posterior determinants.

SCAR/WAVE complex recruitment to a supracellular actomyosin cable by myosin activators and a junctional Arf-GEF during Drosophila dorsal closure.

Expansive Arp2/3 actin networks and contractile actomyosin networks can be spatially and temporally segregated within the cell, but the networks also interact closely at various sites, including adherens junctions. However, molecular mechanisms coordinating these interactions remain unclear. We found that the SCAR/WAVE complex, an Arp2/3 activator, is enriched at adherens junctions of the leading edge actomyosin cable during Drosophila dorsal closure. Myosin activators were both necessary and sufficient for SCAR/WAVE accumulation at leading edge junctions. The same myosin activators were previously shown to recruit the cytohesin Arf-GEF Steppke to these sites, and mammalian studies have linked Arf small G protein signaling to SCAR/WAVE activation. During dorsal closure, we find that Steppke is required for SCAR/WAVE enrichment at the actomyosin-linked junctions. Arp2/3 also localizes to adherens junctions of the leading edge cable. We propose that junctional actomyosin activity acts through Steppke to recruit SCAR/WAVE and Arp2/3 for regulation of the leading edge supracellular actomyosin cable during dorsal closure.

Decisions, decisions: beta-catenin chooses between adhesion and transcription.

beta-catenin functions in both cell adhesion and transcription. Properly choosing between these functions is crucial for normal development, and the wrong choice can lead to cancer. Recent studies have revealed molecular switches that help dictate whether beta-catenin interacts with adhesive or transcriptional complexes. Cara Gottardi and Barry Gumbiner identify Wnt-induced beta-catenin conformational changes that favor assembly into transcription complexes, whereas alpha-catenin-associated beta-catenin appears to favor adhesion. Furthermore, Felix Brembeck and colleagues reveal that phosphorylation dissociates beta-catenin from adhesion complexes while enhancing BCL9-2 binding to promote transcription.

The positioning and segregation of apical cues during epithelial polarity establishment in Drosophila.

Cell polarity is critical for epithelial structure and function. Adherens junctions (AJs) often direct this polarity, but we previously found that Bazooka (Baz) acts upstream of AJs as epithelial polarity is first established in Drosophila. This prompted us to ask how Baz is positioned and how downstream polarity is elaborated. Surprisingly, we found that Baz localizes to an apical domain below its typical binding partners atypical protein kinase C (aPKC) and partitioning defective (PAR)-6 as the Drosophila epithelium first forms. In fact, Baz positioning is independent of aPKC and PAR-6 relying instead on cytoskeletal cues, including an apical scaffold and dynein-mediated basal-to-apical transport. AJ assembly is closely coupled to Baz positioning, whereas aPKC and PAR-6 are positioned separately. This forms a stratified apical domain with Baz and AJs localizing basal to aPKC and PAR-6, and we identify specific mechanisms that keep these proteins apart. These results reveal key steps in the assembly of the apical domain in Drosophila.

The Arf-GEF Steppke promotes F-actin accumulation, cell protrusions and tissue sealing during Drosophila dorsal closure.

Cytohesin Arf-GEFs promote actin polymerization and protrusions of cultured cells, whereas the Drosophila cytohesin, Steppke, antagonizes actomyosin networks in several developmental contexts. To reconcile these findings, we analyzed epidermal leading edge actin networks during Drosophila embryo dorsal closure. Here, Steppke is required for F-actin of the actomyosin cable and for actin-based protrusions. steppke mutant defects in the leading edge actin networks are associated with improper sealing of the dorsal midline, but are distinguishable from effects of myosin mis-regulation. Steppke localizes to leading edge cell-cell junctions with accumulations of the F-actin regulator Enabled emanating from either side. Enabled requires Steppke for full leading edge recruitment, and genetic interaction shows the proteins cooperate for dorsal closure. Inversely, Steppke over-expression induces ectopic, actin-rich, lamellar cell protrusions, an effect dependent on the Arf-GEF activity and PH domain of Steppke, but independent of Steppke recruitment to myosin-rich AJs via its coiled-coil domain. Thus, Steppke promotes actin polymerization and cell protrusions, effects that occur in conjunction with Steppke's previously reported regulation of myosin contractility during dorsal closure.

Adherens junction-dependent and -independent steps in the establishment of epithelial cell polarity in Drosophila.

Adherens junctions (AJs) are thought to be key landmarks for establishing epithelial cell polarity, but the origin of epithelial polarity in Drosophila remains unclear. Thus, we examined epithelial polarity establishment during early Drosophila development. We found apical accumulation of both Drosophila E-Cadherin (DE-Cad) and the apical cue Bazooka (Baz) as cells first form. Mutant analyses revealed that apical Baz accumulations can be established in the absence of AJs, whereas assembly of apical DE-Cad complexes requires Baz. Thus, Baz acts upstream of AJs during epithelial polarity establishment. During gastrulation the absence of AJs results in widespread cell dissociation and depolarization. Some epithelial structures are retained, however. These structures maintain apical Baz, accumulate apical Crumbs, and organize polarized cytoskeletons, but display abnormal cell morphology and fail to segregate the basolateral cue Discs large from the apical domain. Thus, although epithelial polarity develops in the absence of AJs, AJs play specific roles in maintaining epithelial architecture and segregating basolateral cues.

Par-1 controls the composition and growth of cortical actin caps during Drosophila embryo cleavage.

Cell structure depends on the cortex, a thin network of actin polymers and additional proteins underlying the plasma membrane. The cell polarity kinase Par-1 is required for cells to form following syncytial Drosophila embryo development. This requirement stems from Par-1 promoting cortical actin caps that grow into dome-like metaphase compartments for dividing syncytial nuclei. We find the actin caps to be a composite material of Diaphanous (Dia)-based actin bundles interspersed with independently formed, Arp2/3-based actin puncta. Par-1 and Dia colocalize along extended regions of the bundles, and both are required for the bundles and for each other's bundle-like localization, consistent with an actin-dependent self-reinforcement mechanism. Par-1 helps establish or maintain these bundles in a cortical domain with relatively low levels of the canonical formin activator Rho1-GTP. Arp2/3 is required for displacing the bundles away from each other and toward the cap circumference, suggesting interactions between these cytoskeletal components could contribute to the growth of the cap into a metaphase compartment.

Key roles of Arf small G proteins and biosynthetic trafficking for animal development.

Although biosynthetic trafficking can function constitutively, it also functions specifically for certain developmental processes. These processes require either a large increase to biosynthesis or the biosynthesis and targeted trafficking of specific players. We review the conserved molecular mechanisms that direct biosynthetic trafficking, and discuss how their genetic disruption affects animal development. Specifically, we consider Arf small G proteins, such as Arf1 and Sar1, and their coat effectors, COPI and COPII, and how these proteins promote biosynthetic trafficking for cleavage of the Drosophila embryo, the growth of neuronal dendrites and synapses, extracellular matrix secretion for bone development, lumen development in epithelial tubes, notochord and neural tube development, and ciliogenesis. Specific need for the biosynthetic trafficking system is also evident from conserved CrebA/Creb3-like transcription factors increasing the expression of secretory machinery during several of these developmental processes. Moreover, dysfunctional trafficking leads to a range of developmental syndromes.