Tricellular adherens junctions are points of high tension that are central to the rearrangement of epithelial cells. However, the molecular composition of these junctions is unknown, making it difficult to assess their role in morphogenesis. Here, we show that Sidekick, an immunoglobulin family cell adhesion protein, is highly enriched at tricellular adherens junctions in Drosophila. This localization is modulated by tension, and Sidekick is itself necessary to maintain normal levels of cell bond tension. Loss of Sidekick causes defects in cell and junctional rearrangements in actively remodeling epithelial tissues like the retina and tracheal system. The adaptor proteins Polychaetoid and Canoe are enriched at tricellular adherens junctions in a Sidekick-dependent manner; Sidekick functionally interacts with both proteins and directly binds to Polychaetoid. We suggest that Polychaetoid and Canoe link Sidekick to the actin cytoskeleton to enable tricellular adherens junctions to maintain or transmit cell bond tension during epithelial cell rearrangements.

Lattice cells (LCs) in the developing Drosophila retina change shape before attaining final form. Previously, we showed that repeated contraction and expansion of apical cell contacts affect these dynamics. Here, we describe another factor, the assembly of a Rho1-dependent medioapical actomyosin ring formed by nodes linked by filaments that contract the apical cell area. Cell area contraction alternates with relaxation, generating pulsatile changes in cell area that exert force on neighboring LCs. Moreover, Rho1 signaling is sensitive to mechanical changes, becoming active when tension decreases and cells expand, while the negative regulator RhoGAP71E accumulates when tension increases and cells contract. This results in cycles of cell area contraction and relaxation that are reciprocally synchronized between adjacent LCs. Thus, mechanically sensitive Rho1 signaling controls pulsatile medioapical actomyosin contraction and coordinates cell behavior across the epithelium. Disrupting the kinetics of pulsing can lead to developmental errors, suggesting this process controls cell shape and tissue integrity during epithelial morphogenesis of the retina.

Epithelial remodeling of the Drosophila retina depends on the pulsatile contraction and expansion of apical contacts between the cells that form its hexagonal lattice. Phosphoinositide PI(3,4,5)P 3 (PIP 3 ) accumulates around tricellular adherens junctions (tAJs) during contact expansion and dissipates during contraction, but with unknown function. Here we found that manipulations of Pten or Pi3K that either decreased or increased PIP 3 resulted in shortened contacts and a disordered lattice, indicating a requirement for PIP 3 dynamics and turnover. These phenotypes are caused by a loss of protrusive branched actin, resulting from impaired activity of the Rac1 Rho GTPase and the WAVE regulatory complex (WRC). We additionally found that during contact expansion, Pi3K moves into tAJs to promote the cyclical increase of PIP 3 in a spatially and temporally precise manner. Thus, dynamic regulation of PIP 3 by Pten and Pi3K controls the protrusive phase of junctional remodeling, which is essential for planar epithelial morphogenesis.

The Drosophila leg imaginal disc consists of a peripheral region that contributes to adult body wall, and a central region that forms the leg proper. While the patterning signals and transcription factors that determine the identity of adult structures have been identified, the mechanisms that determine the shape of these structures remain largely unknown. The family of Rho GTPases, which consists of seven members in flies, modulates cell adhesion, actomyosin contractility, protrusive membrane activity, and cell-matrix adhesion to generate mechanical forces that shape adult structures. The Rho GTPases are ubiquitously expressed and it remains unclear how they orchestrate morphogenetic events. The Rho guanine nucleotide exchange factors (RhoGEFs) and Rho GTPase activating proteins (RhoGAPs), which respectively activate and deactivate corresponding Rho GTPases, have been proposed to regulate the activity of Rho signaling cascades in specific spatiotemporal patterns to orchestrate morphogenetic events. Here we identify restricted expression of 12 of the 20 RhoGEFs and 10 of the 22 Rho RhoGAPs encoded in Drosophila during metamorphosis. Expression of a subset of each family of RhoGTPase regulators was restricted to motile cell populations including tendon, muscle, trachea, and peripodial stalk cells. A second subset was restricted either to all presumptive joints or only to presumptive tarsal joints. Depletion of individual RhoGEFs and RhoGAPs in the epithelium of the disc proper identified several joint-specific genes, which act downstream of segmental patterning signals to control epithelial morphogenesis. Our studies provide a framework with which to understand how Rho signaling cascades orchestrate complex morphogenetic events in multi-cellular organisms, and evidence that patterning signals regulate these cascades to control apical constriction and epithelial invagination at presumptive joints.

The Drosophila wing imaginal disc is subdivided along the proximodistal axis into the distal pouch, the hinge, the surrounding pleura, and the notum. While the genetic pathways that specify the identity of each of these domains have been well studied, the mechanisms that coordinate the relative expansion of these domains are not well understood. Here we investigated the role of the stat92E signal transducer and activator of transcription in wing proximodistal development. We find that stat92E is active ubiquitously in early wing imaginal discs, where it acts to inhibit the induction of ectopic wing fields. Subsequently, stat92E activity is down regulated in the notum and distal pouch. These dynamics coincide with and contribute to the proportional subdivision and expansion of these primordia. As development proceeds, stat92E activity becomes restricted to the hinge, where it promotes normal expansion of the hinge, and restricts expansion of the notum. We also find that stat92E is required autonomously to specify dorsal pleura identity and inhibit notum identity to properly subdivide the body wall. Our data suggest that stat92E activity is regulated along the proximodistal axis to pattern this axis and control the relative expansion of the pouch, hinge, and notum.

Contractile forces eliminate cell contacts in many morphogenetic processes. However, mechanisms that balance contractile forces to promote subtler remodeling remain unknown. To address this gap, we investigated remodeling of Drosophila eye lattice cells (LCs), which preserve cell contacts as they narrow to form the edges of a multicellular hexagonal lattice. We found that during narrowing, LC-LC contacts dynamically constrict and expand. Similar to other systems, actomyosin-based contractile forces promote pulses of constriction. Conversely, we found that WAVE-dependent branched F-actin accumulates at LC-LC contacts during expansion and functions to expand the cell apical area, promote shape changes, and prevent elimination of LC-LC contacts. Finally, we found that small Rho GTPases regulate the balance of contractile and protrusive dynamics. These data suggest a mechanism by which WAVE regulatory complex-based F-actin dynamics antagonize contractile forces to regulate cell shape and tissue topology during remodeling and thus contribute to the robustness and precision of the process.

The growth and patterning of Drosophila wing and notum primordia depend on their subdivision into progressively smaller domains by secreted signals that emanate from localized sources termed organizers. While the mechanisms that organize the wing primordium have been studied extensively, those that organize the notum are incompletely understood. The genes odd-skipped (odd), drumstick (drm), sob, and bowl comprise the odd-skipped family of C(2)H(2) zinc finger genes, which has been implicated in notum growth and patterning. Here we show that drm, Bowl, and eyegone (eyg), a gene required for notum patterning, accumulate in nested domains in the anterior notum. Ectopic drm organized the nested expression of these anterior notum genes and downregulated the expression of posterior notum genes. The cell-autonomous induction of Bowl and Eyg required bowl, while the non-autonomous effects were independent of bowl. The homeodomain protein Bar is expressed along the anterior border of the notum adjacent to cells expressing the Notch (N) ligand Delta (Dl). bowl was required to promote Bar and repress Dl expression to pattern the anterior notum in a cell-autonomous manner, while lines acted antagonistically to bowl posterior to the Bowl domain. Our data suggest that the odd-skipped genes act at the anterior notum border to organize the notum anterior-posterior (AP) axis using both autonomous and non-autonomous mechanisms.

Contractile actomyosin and protrusive branched F-actin networks interact in a dynamic balance, repeatedly contracting and expanding apical cell contacts to organize the epithelium of the developing fly retina. Previously we showed that the immunoglobulin superfamily protein Sidekick (Sdk) contributes to contraction by recruiting the actin binding protein Polychaetoid (Pyd) to vertices. Here we show that as tension increases during contraction, Sdk progressively accumulates at vertices, where it toggles to recruit the WAVE regulatory complex (WRC) to promote actin branching and protrusion. Sdk alternately interacts with the WRC and Pyd using the same C-terminal motif. With increasing protrusion, levels of Sdk and the WRC decrease at vertices while levels of Pyd increase paving the way for another round of contraction. Thus, by virtue of dynamic association with vertices and interchangeable associations with contractile and protrusive effectors, Sdk is central to controlling the balance between contraction and expansion that shapes this epithelium.

Elongation and invagination of epithelial tissues are fundamental developmental processes that contribute to the morphogenesis of embryonic and adult structures and are dependent on coordinated remodeling of cell-cell contacts. The morphogenesis of Drosophila leg imaginal discs depends on extensive remodeling of cell contacts and thus provides a useful system with which to investigate the underlying mechanisms. The small Rho GTPase regulator RhoGAP68F has been previously implicated in leg morphogenesis. It consists of on an N-terminal Sec14 domain and a C-terminal GAP domain. Here we examined the molecular function and role of RhoGAP68F in epithelial remodeling. We find that depletion of RhoGAP68F impairs epithelial remodeling from a pseudostratified to simple, while overexpression of RhoGAP68F causes tears of lateral cell-cell contacts and thus impairs epithelial integrity. We show that the RhoGAP68F protein localizes to Rab4 recycling endosomes and forms a complex with the Rab4 protein. The Sec14 domain is sufficient for localizing to Rab4 endosomes, while the activity of the GAP domain is dispensable. RhoGAP68F, in turn, inhibits the scission and movement of Rab4 endosomes involved in transport the adhesion proteins Fasciclin3 and E-cadherin back to cell-cell contacts. Expression of RhoGAP68F is upregulated during prepupal development suggesting that RhoGAP68F decreases the transport of key adhesion proteins to the cell surface during this developmental stage to decrease the strength of adhesive cell-cell contacts and thereby facilitate epithelial remodeling and leg morphogenesis.