Fat2 polarizes Lar and Sema5c to coordinate the motility of collectively migrating epithelial cells.

Migrating epithelial cells globally align their migration machinery to achieve tissue-level movement. Biochemical signaling across leading-trailing cell-cell interfaces can promote this alignment by partitioning migratory behaviors like protrusion and retraction to opposite sides of the interface. However, how signaling proteins become organized at interfaces to accomplish this is poorly understood. The follicular epithelial cells of Drosophila melanogaster have two signaling modules at their leading-trailing interfaces - one composed of the atypical cadherin Fat2 (also known as Kugelei) and the receptor tyrosine phosphatase Lar, and one composed of Semaphorin5c and its receptor Plexin A. Here, we show that these modules form one interface signaling system with Fat2 at its core. Trailing edge-enriched Fat2 concentrates both Lar and Semaphorin5c at leading edges of cells, but Lar and Semaphorin5c play little role in the localization of Fat2. Fat2 is also more stable at interfaces than Lar or Semaphorin5c. Once localized, Lar and Semaphorin5c act in parallel to promote collective migration. We propose that Fat2 serves as the organizer of this interface signaling system by coupling and polarizing the distributions of multiple effectors that work together to align the migration machinery of neighboring cells.

Oriented basement membrane fibrils provide a memory for F-actin planar polarization via the Dystrophin-Dystroglycan complex during tissue elongation.

How extracellular matrix contributes to tissue morphogenesis is still an open question. In the Drosophila ovarian follicle, it has been proposed that after Fat2-dependent planar polarization of the follicle cell basal domain, oriented basement membrane (BM) fibrils and F-actin stress fibers constrain follicle growth, promoting its axial elongation. However, the relationship between BM fibrils and stress fibers and their respective impact on elongation are unclear. We found that Dystroglycan (Dg) and Dystrophin (Dys) are involved in BM fibril deposition. Moreover, they also orient stress fibers, by acting locally and in parallel to Fat2. Importantly, Dg-Dys complex-mediated cell-autonomous control of F-actin fiber orientation relies on the preceding BM fibril deposition, indicating two distinct but interdependent functions. Thus, the Dg-Dys complex works as a crucial organizer of the epithelial basal domain, regulating both F-actin and BM. Furthermore, BM fibrils act as a persistent cue for the orientation of stress fibers that are the main effector of elongation.

The dPix-Git complex is essential to coordinate epithelial morphogenesis and regulate myosin during Drosophila egg chamber development.

How biochemical and mechanical information are integrated during tissue development is a central question in morphogenesis. In many biological systems, the PIX-GIT complex localises to focal adhesions and integrates both physical and chemical information. We used Drosophila melanogaster egg chamber formation to study the function of PIX and GIT orthologues (dPix and Git, respectively), and discovered a central role for this complex in controlling myosin activity and epithelial monolayering. We found that Git's focal adhesion targeting domain mediates basal localisation of this complex to filament structures and the leading edge of migrating cells. In the absence of dpix and git, tissue disruption is driven by contractile forces, as reduction of myosin activators restores egg production and morphology. Further, dpix and git mutant eggs closely phenocopy defects previously reported in pak mutant epithelia. Together, these results indicate that the dPix-Git complex controls egg chamber morphogenesis by controlling myosin contractility and Pak kinase downstream of focal adhesions.

Influence of ovarian muscle contraction and oocyte growth on egg chamber elongation in Drosophila.

Organs are formed from multiple cell types that make distinct contributions to their shape. The Drosophila egg chamber provides a tractable model to dissect such contributions during morphogenesis. Egg chambers consist of 16 germ cells (GCs) surrounded by a somatic epithelium. Initially spherical, these structures elongate as they mature. This morphogenesis is thought to occur through a 'molecular corset' mechanism, whereby structural elements within the epithelium become circumferentially organized perpendicular to the elongation axis and resist the expansive growth of the GCs to promote elongation. Whether this epithelial organization provides the hypothesized constraining force has been difficult to discern, however, and a role for GC growth has not been demonstrated. Here, we provide evidence for this mechanism by altering the contractile activity of the tubular muscle sheath that surrounds developing egg chambers. Muscle hypo-contraction indirectly reduces GC growth and shortens the egg, which demonstrates the necessity of GC growth for elongation. Conversely, muscle hyper-contraction enhances the elongation program. Although this is an abnormal function for this muscle, this observation suggests that a corset-like force from the egg chamber's exterior could promote its lengthening. These findings highlight how physical contributions from several cell types are integrated to shape an organ.

Dynamic regulation of basement membrane protein levels promotes egg chamber elongation in Drosophila.

Basement membranes (BMs) are sheet-like extracellular matrices that provide essential support to epithelial tissues. Recent evidence suggests that regulated changes in BM architecture can direct tissue morphogenesis, but the mechanisms by which cells remodel BMs are largely unknown. The Drosophila egg chamber is an organ-like structure that transforms from a spherical to an ellipsoidal shape as it matures. This elongation coincides with a stage-specific increase in Type IV Collagen (Col IV) levels in the BM surrounding the egg chamber; however, the mechanisms and morphogenetic relevance of this remodeling event have not been established. Here, we identify the Collagen-binding protein SPARC as a negative regulator of egg chamber elongation, and show that SPARC down-regulation is necessary for the increase in Col IV levels to occur. We find that SPARC interacts with Col IV prior to secretion and propose that, through this interaction, SPARC blocks the incorporation of newly synthesized Col IV into the BM. We additionally observe a decrease in Perlecan levels during elongation, and show that Perlecan is a negative regulator of this process. These data provide mechanistic insight into SPARC's conserved role in matrix dynamics and demonstrate that regulated changes in BM composition influence organ morphogenesis.

Building from the Ground up: Basement Membranes in Drosophila Development.

Basement membranes (BMs) are sheetlike extracellular matrices found at the basal surfaces of epithelial tissues. The structural and functional diversity of these matrices within the body endows them with the ability to affect multiple aspects of cell behavior and communication; for this reason, BMs are integral to many developmental processes. The power of Drosophila genetics, as applied to the BM, has yielded substantial insight into how these matrices influence development. Here, we explore three facets of BM biology to which Drosophila research has made particularly important contributions. First, we discuss how newly synthesized BM proteins are secreted to and assembled exclusively on basal epithelial surfaces. Next, we examine how regulation of the structural properties of the BM mechanically supports and guides tissue morphogenesis. Finally, we explore how BMs influence development through the modulation of several major signaling pathways.

Cortical tension promotes Kibra degradation via Par-1.

The Hippo pathway is an evolutionarily conserved regulator of tissue growth. Multiple Hippo signaling components are regulated via proteolytic degradation. However, how these degradation mechanisms are themselves modulated remains unexplored. Kibra is a key upstream pathway activator that promotes its own ubiquitin-mediated degradation upon assembling a Hippo signaling complex. Here, we demonstrate that Hippo complex-dependent Kibra degradation is modulated by cortical tension. Using classical genetic, osmotic, and pharmacological manipulations of myosin activity and cortical tension, we show that increasing cortical tension leads to Kibra degradation, whereas decreasing cortical tension increases Kibra abundance. Our study also implicates Par-1 in regulating Kib abundance downstream of cortical tension. We demonstrate that Par-1 promotes ubiquitin-mediated Kib degradation in a Hippo complex-dependent manner and is required for tension-induced Kib degradation. Collectively, our results reveal a previously unknown molecular mechanism by which cortical tension affects Hippo signaling and provide novel insights into the role of mechanical forces in growth control.

Fat2 polarizes Lar and Sema5c to coordinate the motility of collectively migrating epithelial cells.

Migrating epithelial cells globally align their migration machinery to achieve tissue-level movement. Biochemical signaling across leading-trailing cell-cell interfaces can promote this alignment by partitioning migratory behaviors like protrusion and retraction to opposite sides of the interface. However, how the necessary signaling proteins become organized at this site is poorly understood. The follicular epithelial cells of Drosophila melanogaster have two signaling modules at their leading-trailing interfaces-one composed of the atypical cadherin Fat2 and the receptor tyrosine phosphatase Lar, and one composed of Semaphorin 5c and its receptor Plexin A. Here we show that these modules form one interface signaling system with Fat2 at its core. Trailing edge-enriched Fat2 concentrates both Lar and Sema5c at cells' leading edges, likely by slowing their turnover at this site. Once localized, Lar and Sema5c act in parallel to promote collective migration. Our data suggest a model in which Fat2 couples and polarizes the distributions of multiple effectors that work together to align the migration machinery of neighboring cells.

Optimized Fixation and Phalloidin Staining of Basally Localized F-Actin Networks in Collectively Migrating Follicle Cells.

The follicular epithelial cells of the Drosophila egg chamber have become a premier model to study how cells globally orient their actin-based machinery for collective migration. The basal surface of each follicle cell has lamellipodial and filopodial protrusions that extend from its leading edge and an array of stress fibers that mediate its adhesion to the extracellular matrix; these migratory structures are all globally aligned in the direction of tissue movement. To understand how this global alignment is achieved, one must be able to reliably visualize the underlying F-actin; however, dynamic F-actin networks can be difficult to preserve in fixed tissues. Here, we describe an optimized protocol for the fixation and phalloidin staining of the follicular epithelium. We also provide a brief primer on relevant aspects of the image acquisition process to ensure high quality data are collected.

A Low-Tech Flow Chamber for Live Imaging of Drosophila Egg Chambers During Drug Treatments.

The Drosophila egg chamber is a powerful system to study epithelial cell collective migration and polarized basement membrane secretion. A strength of this system is the ability to capture these dynamic processes in ex vivo organ culture using high-resolution live imaging. Ex vivo culture also allows acute pharmacological or labeling treatments, extending the versatility of the system. However, many current ex vivo egg chamber culture setups do not permit easy medium exchange, preventing researchers from following individual egg chambers through multiple treatments. Here we present a method to immobilize egg chambers in an easy-to-construct flow chamber that permits imaging of the same egg chamber through repeated solution exchanges. This will allow researchers to take greater advantage of the wide variety of available pharmacological perturbations and other treatments like dyes to study dynamic processes in the egg chamber.

Kinesin-directed secretion of basement membrane proteins to a subdomain of the basolateral surface in Drosophila epithelial cells.

Epithelial tissues are lined with a sheet-like basement membrane (BM) extracellular matrix at their basal surfaces that plays essential roles in adhesion and signaling. BMs also provide mechanical support to guide morphogenesis. Despite their importance, we know little about how epithelial cells secrete and assemble BMs during development. BM proteins are sorted into a basolateral secretory pathway distinct from other basolateral proteins. Because BM proteins self-assemble into networks, and the BM lines only a small portion of the basolateral domain, we hypothesized that the site of BM protein secretion might be tightly controlled. Using the Drosophila follicular epithelium, we show that kinesin-3 and kinesin-1 motors work together to define this secretion site. Similar to all epithelia, the follicle cells have polarized microtubules (MTs) along their apical-basal axes. These cells collectively migrate, and they also have polarized MTs along the migratory axis at their basal surfaces. We find follicle cell MTs form one interconnected network, which allows kinesins to transport Rab10+ BM secretory vesicles both basally and to the trailing edge of each cell. This positions them near the basal surface and the basal-most region of the lateral domain for exocytosis. When kinesin transport is disrupted, the site of BM protein secretion is expanded, and ectopic BM networks form between cells that impede migration and disrupt tissue architecture. These results show how epithelial cells can define a subdomain on their basolateral surface through MT-based transport and highlight the importance of controlling the exocytic site of network-forming proteins.

Fat2 polarizes the WAVE complex in trans to align cell protrusions for collective migration.

For a group of cells to migrate together, each cell must couple the polarity of its migratory machinery with that of the other cells in the cohort. Although collective cell migrations are common in animal development, little is known about how protrusions are coherently polarized among groups of migrating epithelial cells. We address this problem in the collective migration of the follicular epithelial cells in Drosophila melanogaster. In this epithelium, the cadherin Fat2 localizes to the trailing edge of each cell and promotes the formation of F-actin-rich protrusions at the leading edge of the cell behind. We show that Fat2 performs this function by acting in trans to concentrate the activity of the WASP family verprolin homolog regulatory complex (WAVE complex) at one long-lived region along each cell's leading edge. Without Fat2, the WAVE complex distribution expands around the cell perimeter and fluctuates over time, and protrusive activity is reduced and unpolarized. We further show that Fat2's influence is very local, with sub-micron-scale puncta of Fat2 enriching the WAVE complex in corresponding puncta just across the leading-trailing cell-cell interface. These findings demonstrate that a trans interaction between Fat2 and the WAVE complex creates stable regions of protrusive activity in each cell and aligns the cells' protrusions across the epithelium for directionally persistent collective migration.

Dynein regulates epithelial polarity and the apical localization of stardust A mRNA.

Intense investigation has identified an elaborate protein network controlling epithelial polarity. Although precise subcellular targeting of apical and basolateral determinants is required for epithelial architecture, little is known about how the individual determinant proteins become localized within the cell. Through a genetic screen for epithelial defects in the Drosophila follicle cells, we have found that the cytoplasmic Dynein motor is an essential regulator of apico-basal polarity. Our data suggest that Dynein acts through the cytoplasmic scaffolding protein Stardust (Sdt) to localize the transmembrane protein Crumbs, in part through the apical targeting of specific sdt mRNA isoforms. We have mapped the sdt mRNA localization signal to an alternatively spliced coding exon. Intriguingly, the presence or absence of this exon corresponds to a developmental switch in sdt mRNA localization in which apical transcripts are only found during early stages of epithelial development, while unlocalized transcripts predominate in mature epithelia. This work represents the first demonstration that Dynein is required for epithelial polarity and suggests that mRNA localization may have a functional role in the regulation of apico-basal organization. Moreover, we introduce a unique mechanism in which alternative splicing of a coding exon is used to control mRNA localization during development.

DAAM mediates the assembly of long-lived, treadmilling stress fibers in collectively migrating epithelial cells in Drosophila.

Stress fibers (SFs) are actomyosin bundles commonly found in individually migrating cells in culture. However, whether and how cells use SFs to migrate in vivo or collectively is largely unknown. Studying the collective migration of the follicular epithelial cells in Drosophila, we found that the SFs in these cells show a novel treadmilling behavior that allows them to persist as the cells migrate over multiple cell lengths. Treadmilling SFs grow at their fronts by adding new integrin-based adhesions and actomyosin segments over time. This causes the SFs to have many internal adhesions along their lengths, instead of adhesions only at the ends. The front-forming adhesions remain stationary relative to the substrate and typically disassemble as the cell rear approaches. By contrast, a different type of adhesion forms at the SF's terminus that slides with the cell's trailing edge as the actomyosin ahead of it shortens. We further show that SF treadmilling depends on cell movement and identify a developmental switch in the formins that mediate SF assembly, with Dishevelled-associated activator of morphogenesis acting during migratory stages and Diaphanous acting during postmigratory stages. We propose that treadmilling SFs keep each cell on a linear trajectory, thereby promoting the collective motility required for epithelial migration.

Mobilizing the Matrix for Organ Morphogenesis.

How a basement membrane continuously surrounds an organ that is growing and changing shape is not yet understood. In this issue of Developmental Cell, Matsubayashi et al. and Keeley et al. address this question by showing that individual basement membrane proteins are more dynamic than previously thought.

The Drosophila micropyle as a system to study how epithelia build complex extracellular structures.

Dynamic rearrangements of epithelial cells play central roles in shaping tissues and organs during development. There are also scenarios, however, in which epithelial cell movements synergize with the secretion of extracellular matrix to build rigid, acellular structures that persist long after the cells are gone. The formation of the Drosophila micropyle provides an elegant example of this epithelial craftsmanship. The micropyle is a cone-shaped projection of the eggshell through which the sperm will enter to fertilize the oocyte. Though simple on the surface, both the inner structure and construction of the micropyle are remarkably complex. In this review, I first provide an overview of egg development, focusing on the key events required to understand micropyle formation. I then describe the structure of the micropyle, the cellular contributions to its morphogenesis and some interesting open questions about this process. There is a brief discussion of micropyle formation in other insects and fish to highlight the potential for comparative studies. Finally, I discuss how new studies of micropyle formation could reveal general mechanisms that epithelia use to build complex extracellular structures. This article is part of a discussion meeting issue 'Contemporary morphogenesis'.

ESCargo: a regulatable fluorescent secretory cargo for diverse model organisms.

Membrane traffic can be studied by imaging a cargo protein as it transits the secretory pathway. The best tools for this purpose initially block export of the secretory cargo from the endoplasmic reticulum (ER) and then release the block to generate a cargo wave. However, previously developed regulatable secretory cargoes are often tricky to use or specific for a single model organism. To overcome these hurdles for budding yeast, we recently optimized an artificial fluorescent secretory protein that exits the ER with the aid of the Erv29 cargo receptor, which is homologous to mammalian Surf4. The fluorescent secretory protein forms aggregates in the ER lumen and can be rapidly disaggregated by addition of a ligand to generate a nearly synchronized cargo wave. Here we term this regulatable secretory protein ESCargo (Erv29/Surf4-dependent secretory cargo) and demonstrate its utility not only in yeast cells, but also in cultured mammalian cells, Drosophila cells, and the ciliate Tetrahymena thermophila. Kinetic studies indicate that rapid export from the ER requires recognition by Erv29/Surf4. By choosing an appropriate ER signal sequence and expression vector, this simple technology can likely be used with many model organisms.

In-silico definition of the Drosophila melanogaster matrisome.

The extracellular matrix (ECM) is an assembly of hundreds of proteins that structurally supports the cells it surrounds and biochemically regulates their functions. Drosophila melanogaster has emerged as a powerful model organism to study fundamental mechanisms underlying ECM protein secretion, ECM assembly, and ECM roles in pathophysiological processes. However, as of today, we do not possess a well-defined list of the components forming the ECM of this organism. We previously reported the development of computational pipelines to define the matrisome - the ensemble of genes encoding ECM and ECM-associated proteins - of humans, mice, zebrafish and C. elegans. Using a similar approach, we report here that our pipeline has identified 641 genes constituting the Drosophila matrisome. We further classify these genes into different structural and functional categories, including an expanded way to classify genes encoding proteins forming apical ECMs. We illustrate how having a comprehensive list of Drosophila matrisome proteins can be used to annotate large proteomic datasets and identify unsuspected roles for the ECM in pathophysiological processes. Last, to aid the dissemination and usage of the proposed definition and categorization of the Drosophila matrisome by the scientific community, our list has been made available through three public portals: The Matrisome Project (http://matrisome.org), The FlyBase (https://flybase.org/), and GLAD (https://www.flyrnai.org/tools/glad/web/).

Planar-Polarized Semaphorin-5c and Plexin A Promote the Collective Migration of Epithelial Cells in Drosophila.

Collective migration of epithelial cells is essential for morphogenesis, wound repair, and the spread of many cancers, yet how individual cells signal to one another to coordinate their movements is largely unknown. Here, we introduce a tissue-autonomous paradigm for semaphorin-based regulation of collective cell migration. Semaphorins typically regulate the motility of neuronal growth cones and other migrating cell types by acting as repulsive cues within the migratory environment. Studying the follicular epithelial cells of Drosophila, we discovered that the transmembrane semaphorin, Sema-5c, promotes collective cell migration by acting within the migrating cells themselves, not the surrounding environment. Sema-5c is planar polarized at the basal epithelial surface such that it is enriched at the leading edge of each cell. This location places it in a prime position to send a repulsive signal to the trailing edge of the cell ahead to communicate directional information between neighboring cells. Our data show that Sema-5c can signal across cell-cell boundaries to suppress protrusions in neighboring cells and that Plexin A is the receptor that transduces this signal. Finally, we present evidence that Sema-5c antagonizes the activity of Lar, another transmembrane guidance cue that operates along leading-trailing cell-cell interfaces in this tissue, via a mechanism that appears to be independent of Plexin A. Together, our results suggest that multiple transmembrane guidance cues can be deployed in a planar-polarized manner across an epithelium and work in concert to coordinate individual cell movements for collective migration.