AdamTS proteases control basement membrane heterogeneity and organ shape in Drosophila.

The basement membrane (BM) is an extracellular matrix that plays important roles in animal development. A spatial heterogeneity in composition and structural properties of the BM provide cells with vital cues for morphogenetic processes such as cell migration or cell polarization. Here, using the Drosophila egg chamber as a model system, we show that the BM becomes heterogeneous during development, with a reduction in Collagen IV density at the posterior pole and differences in the micropattern of aligned fiber-like structures. We identified two AdamTS matrix proteases required for the proper elongated shape of the egg chamber, yet the molecular mechanisms by which they act are different. Stall is required to establish BM heterogeneity by locally limiting Collagen IV protein density, whereas AdamTS-A alters the micropattern of fiber-like structures within the BM at the posterior pole. Our results suggest that AdamTS proteases control BM heterogeneity required for organ shape.

A cellular tilting mechanism important for dynamic tissue shape changes and cell differentiation in Drosophila.

Dynamic changes in three-dimensional cell shape are important for tissue form and function. In the developing Drosophila eye, photoreceptor differentiation requires the progression across the tissue of an epithelial fold known as the morphogenetic furrow. Morphogenetic furrow progression involves apical cell constriction and movement of apical cell edges. Here, we show that cells progressing through the morphogenetic furrow move their basal edges in opposite direction to their apical edges, resulting in a cellular tilting movement. We further demonstrate that cells generate, at their basal side, oriented, force-generating protrusions. Knockdown of the protein kinase Src42A or photoactivation of a dominant-negative form of the small GTPase Rac1 reduces protrusion formation. Impaired protrusion formation stalls basal cell movement and slows down morphogenetic furrow progression and photoreceptor differentiation. This work identifies a cellular tilting mechanism important for the generation of dynamic tissue shape changes and cell differentiation.

Hedgehog morphogen gradient is robust towards variations in tissue morphology in Drosophila.

During tissue development, gradients of secreted signaling molecules known as morphogens provide cells with positional information. The mechanisms underlying morphogen spreading have been widely studied, however, it remains largely unexplored whether the shape of morphogen gradients is influenced by tissue morphology. Here, we developed an analysis pipeline to quantify the distribution of proteins within a curved tissue. We applied it to the Hedgehog morphogen gradient in the Drosophila wing and eye-antennal imaginal discs, which are flat and curved tissues, respectively. Despite a different expression profile, the slope of the Hedgehog gradient was comparable between the two tissues. Moreover, inducing ectopic folds in wing imaginal discs did not affect the slope of the Hedgehog gradient. Suppressing curvature in the eye-antennal imaginal disc also did not alter the Hedgehog gradient slope but led to ectopic Hedgehog expression. In conclusion, through the development of an analysis pipeline that allows quantifying protein distribution in curved tissues, we show that the Hedgehog gradient is robust towards variations in tissue morphology.

Preferential recruitment and stabilization of Myosin II at compartment boundaries in Drosophila.

The regulation of mechanical tension exerted at cell junctions guides cell behavior during tissue formation and homeostasis. Cell junctions along compartment boundaries, which are lineage restrictions separating cells with different fates and functions within tissues, are characterized by increased mechanical tension compared to that of cell junctions in the bulk of the tissue. Mechanical tension depends on the actomyosin cytoskeleton; however, the mechanisms by which mechanical tension is locally increased at cell junctions along compartment boundaries remain elusive. Here, we show that non-muscle Myosin II and F-actin transiently accumulate and mechanical tension is increased at cell junctions along the forming anteroposterior compartment boundary in the Drosophila melanogaster pupal abdominal epidermis. Fluorescence recovery after photobleaching experiments showed that Myosin II accumulation correlated with its increased stabilization at these junctions. Moreover, photoconversion experiments indicated that Myosin II is preferentially recruited within cells to junctions along the compartment boundary. Our results indicate that the preferential recruitment and stabilization of Myosin II contribute to the initial build-up of mechanical tension at compartment boundaries.

Distinct contributions of ECM proteins to basement membrane mechanical properties in Drosophila.

The basement membrane is a specialized extracellular matrix (ECM) that is crucial for the development of epithelial tissues and organs. In Drosophila, the mechanical properties of the basement membrane play an important role in the proper elongation of the developing egg chamber; however, the molecular mechanisms contributing to basement membrane mechanical properties are not fully understood. Here, we systematically analyze the contributions of individual ECM components towards the molecular composition and mechanical properties of the basement membrane underlying the follicle epithelium of Drosophila egg chambers. We find that the Laminin and Collagen IV networks largely persist in the absence of the other components. Moreover, we show that Perlecan and Collagen IV, but not Laminin or Nidogen, contribute greatly towards egg chamber elongation. Similarly, Perlecan and Collagen, but not Laminin or Nidogen, contribute towards the resistance of egg chambers against osmotic stress. Finally, using atomic force microscopy we show that basement membrane stiffness mainly depends on Collagen IV. Our analysis reveals how single ECM components contribute to the mechanical properties of the basement membrane controlling tissue and organ shape.

Charting the unknown currents of cellular flows and forces.

One of the central questions in developmental biology concerns how cells become organized into tissues of the correct size, shape and polarity. This organization depends on the implementation of a cell's genetic information to give rise to specific and coordinated cell behaviors, including cell division and cell shape change. The execution of these cell behaviors requires the active generation of mechanical forces. However, understanding how force generation is controlled and, importantly, coordinated among many cells in a tissue was little explored until the early 2000s. Suzanne Eaton was one of the pioneers in this emerging field of developmental tissue mechanics. As we briefly review here, she connected the quantitative analysis of cell behaviors with genetic assays, and integrated physical modeling with measurements of mechanical forces to reveal fundamental insights into epithelial morphogenesis at cell- and tissue-level scales.

Increased lateral tension is sufficient for epithelial folding in Drosophila.

The folding of epithelial sheets is important for tissues, organs and embryos to attain their proper shapes. Epithelial folding requires subcellular modulations of mechanical forces in cells. Fold formation has mainly been attributed to mechanical force generation at apical cell sides, but several studies indicate a role of mechanical tension at lateral cell sides in this process. However, whether lateral tension increase is sufficient to drive epithelial folding remains unclear. Here, we have used optogenetics to locally increase mechanical force generation at apical, lateral or basal sides of epithelial Drosophila wing disc cells, an important model for studying morphogenesis. We show that optogenetic recruitment of RhoGEF2 to apical, lateral or basal cell sides leads to local accumulation of F-actin and increase in mechanical tension. Increased lateral tension, but not increased apical or basal tension, results in sizeable fold formation. Our results stress the diversification of folding mechanisms between different tissues and highlight the importance of lateral tension increase for epithelial folding.

Establishing compartment boundaries in Drosophila wing imaginal discs: An interplay between selector genes, signaling pathways and cell mechanics.

The partitioning of cells into groups or 'compartments' separated by straight and sharp boundaries is important for tissue formation in animal development. Cells from neighboring compartments are characterized by distinct fates and functions and their continuous separation at compartment boundaries maintains proper tissue organization. Signaling across compartment boundaries can induce the local expression of morphogens that in turn direct growth and patterning of the surrounding cells. Compartment boundaries play therefore an important role in tissue development. Compartment boundaries were first identified in the early 1970s in the Drosophila wing. Here, we review the role of compartment boundaries in growth and patterning of the developing wing and then discuss the genetic and physical mechanisms underlying cell separation at compartment boundaries in this tissue.

Tissue mechanical properties modulate cell extrusion in the Drosophila abdominal epidermis.

The replacement of cells is a common strategy during animal development. In the Drosophila pupal abdomen, larval epidermal cells (LECs) are replaced by adult progenitor cells (histoblasts). Previous work showed that interactions between histoblasts and LECs result in apoptotic extrusion of LECs during early pupal development. Extrusion of cells is closely preceded by caspase activation and is executed by contraction of a cortical actomyosin cable. Here, we identify a population of LECs that extrudes independently of the presence of histoblasts during late pupal development. Extrusion of these LECs is not closely preceded by caspase activation, involves a pulsatile medial actomyosin network, and correlates with a developmental time period when mechanical tension and E-cadherin turnover at adherens junctions is particularly high. Our work reveals a developmental switch in the cell extrusion mechanism that correlates with changes in tissue mechanical properties.

Wingless counteracts epithelial folding by increasing mechanical tension at basal cell edges in Drosophila.

The modulation of mechanical tension is important for sculpturing tissues during animal development, yet how mechanical tension is controlled remains poorly understood. In Drosophila wing discs, the local reduction of mechanical tension at basal cell edges results in basal relaxation and the formation of an epithelial fold. Here, we show that Wingless, which is expressed next to this fold, promotes basal cell edge tension to suppress the formation of this fold. Ectopic expression of Wingless blocks fold formation, whereas the depletion of Wingless increases fold depth. Moreover, local depletion of Wingless in a region where Wingless signal transduction is normally high results in ectopic fold formation. The depletion of Wingless also results in decreased basal cell edge tension and basal cell area relaxation. Conversely, the activation of Wingless signal transduction leads to increased basal cell edge tension and basal cell area constriction. Our results identify the Wingless signal transduction pathway as a crucial modulator of mechanical tension that is important for proper wing disc morphogenesis.

Differential lateral and basal tension drive folding of Drosophila wing discs through two distinct mechanisms.

Epithelial folding transforms simple sheets of cells into complex three-dimensional tissues and organs during animal development. Epithelial folding has mainly been attributed to mechanical forces generated by an apically localized actomyosin network, however, contributions of forces generated at basal and lateral cell surfaces remain largely unknown. Here we show that a local decrease of basal tension and an increased lateral tension, but not apical constriction, drive the formation of two neighboring folds in developing Drosophila wing imaginal discs. Spatially defined reduction of extracellular matrix density results in local decrease of basal tension in the first fold; fluctuations in F-actin lead to increased lateral tension in the second fold. Simulations using a 3D vertex model show that the two distinct mechanisms can drive epithelial folding. Our combination of lateral and basal tension measurements with a mechanical tissue model reveals how simple modulations of surface and edge tension drive complex three-dimensional morphological changes.

The Selector Gene apterous and Notch Are Required to Locally Increase Mechanical Cell Bond Tension at the Drosophila Dorsoventral Compartment Boundary.

The separation of cells with distinct fates and functions is important for tissue and organ formation during animal development. Regions of different fates within tissues are often separated from another along straight boundaries. These compartment boundaries play a crucial role in tissue patterning and growth by stably positioning organizers. In Drosophila, the wing imaginal disc is subdivided into a dorsal and a ventral compartment. Cells of the dorsal, but not ventral, compartment express the selector gene apterous. Apterous expression sets in motion a gene regulatory cascade that leads to the activation of Notch signaling in a few cell rows on either side of the dorsoventral compartment boundary. Both Notch and apterous mutant clones disturb the separation of dorsal and ventral cells. Maintenance of the straight shape of the dorsoventral boundary involves a local increase in mechanical tension at cell bonds along the boundary. The mechanisms by which cell bond tension is locally increased however remain unknown. Here we use a combination of laser ablation of cell bonds, quantitative image analysis, and genetic mutants to show that Notch and Apterous are required to increase cell bond tension along the dorsoventral compartment boundary. Moreover, clonal expression of the Apterous target gene capricious results in cell separation and increased cell bond tension at the clone borders. Finally, using a vertex model to simulate tissue growth, we find that an increase in cell bond tension at the borders of cell clones, but not throughout the cell clone, can lead to cell separation. We conclude that Apterous and Notch maintain the characteristic straight shape of the dorsoventral compartment boundary by locally increasing cell bond tension.

Regulating mechanical tension at compartment boundaries in Drosophila.

During animal development, cells with similar function and fate often stay together and sort out from cells with different fates. In Drosophila wing imaginal discs, cells of anterior and posterior fates are separated by a straight compartment boundary. Separation of anterior and posterior cells requires the homeodomain-containing protein Engrailed, which is expressed in posterior cells. Engrailed induces the expression of the short-range signaling molecule Hedgehog in posterior cells and confines Hedgehog signal transduction to anterior cells. Transduction of the Hedgehog signal in anterior cells is required for the separation of anterior and posterior cells. Previous work showed that this separation of cells involves a local increase in mechanical tension at cell junctions along the compartment boundary. However, how mechanical tension was locally increased along the compartment boundary remained unknown. A recent paper now shows that the difference in Hedgehog signal transduction between anterior and posterior cells is necessary and sufficient to increase mechanical tension. The local increase in mechanical tension biases junctional rearrangements during cell intercalations to maintain the straight shape of the compartment boundary. These data highlight how developmental signals can generate patterns of mechanical tension important for tissue organization.

A Mutation in fat2 Uncouples Tissue Elongation from Global Tissue Rotation.

Global tissue rotation was proposed as a morphogenetic mechanism controlling tissue elongation. In Drosophila ovaries, global tissue rotation of egg chambers coincides with egg chamber elongation. Egg chamber rotation was put forward to result in circumferential alignment of extracellular fibers. These fibers serve as molecular corsets to restrain growth of egg chambers perpendicular to the anteroposterior axis, thereby leading to the preferential egg chamber elongation along this axis. The atypical cadherin Fat2 is required for egg chamber elongation, rotation, and the circumferential alignment of extracellular fibers. Here, we have generated a truncated form of Fat2 that lacks the entire intracellular region. fat2 mutant egg chambers expressing this truncated protein fail to rotate yet display normal extracellular fiber alignment and properly elongate. Our data suggest that global tissue rotation, even though coinciding with tissue elongation, is not a necessary prerequisite for elongation.

Memorizing Shape to Orient Cell Division.

A century ago, Oscar Hertwig discovered that cells orient their cleavage plane orthogonal to their long axis. Reporting recently in Nature, Bosveld et al. (2016) shed light on how, showing that NuMA/Mud localization at tricellular junctions provides mitotic cells with the memory of interphase shape used to orient cleavage plane.

A local difference in Hedgehog signal transduction increases mechanical cell bond tension and biases cell intercalations along the Drosophila anteroposterior compartment boundary.

Tissue organization requires the interplay between biochemical signaling and cellular force generation. The formation of straight boundaries separating cells with different fates into compartments is important for growth and patterning during tissue development. In the developing Drosophila wing disc, maintenance of the straight anteroposterior (AP) compartment boundary involves a local increase in mechanical tension at cell bonds along the boundary. The biochemical signals that regulate mechanical tension along the AP boundary, however, remain unknown. Here, we show that a local difference in Hedgehog signal transduction activity between anterior and posterior cells is necessary and sufficient to increase mechanical tension along the AP boundary. This difference in Hedgehog signal transduction is also required to bias cell rearrangements during cell intercalations to keep the characteristic straight shape of the AP boundary. Moreover, severing cell bonds along the AP boundary does not reduce tension at neighboring bonds, implying that active mechanical tension is upregulated, cell bond by cell bond. Finally, differences in the expression of the homeodomain-containing protein Engrailed also contribute to the straight shape of the AP boundary, independently of Hedgehog signal transduction and without modulating cell bond tension. Our data reveal a novel link between local differences in Hedgehog signal transduction and a local increase in active mechanical tension of cell bonds that biases junctional rearrangements. The large-scale shape of the AP boundary thus emerges from biochemical signals inducing patterns of active tension on cell bonds.

Signals and mechanics shaping compartment boundaries in Drosophila.

During animal development groups of cells with similar fates and functions often stay together and separate from cells with different fates. An example for this cellular behavior is the formation of compartments, groups of cells with similar fates that are separated by sharp boundaries from neighboring groups of cells. Compartments play important roles during patterning by serving as units of growth and gene expression. Boundaries between compartments are associated with organizers that secrete signaling molecules instructing growth and differentiation throughout the tissue. The straight shape of the boundary between compartments is important for maintaining the position and shape of the organizer and thus for precise patterning. The straight shape of compartment boundaries, however, is challenged by cell divisions and cell intercalations that take place in many developing tissues. Early work established a role for selector genes and signaling pathways in setting up and keeping boundaries straight. Recent work in Drosophila has now begun to further unravel the physical and cellular mechanisms that maintain compartment boundaries. Key to the separation of compartments is a local increase of actomyosin-dependent mechanical tension at cell junctions along the boundary. Increased mechanical tension acts as a barrier to cell mixing during cell division and influences cell rearrangements during cell intercalations along the compartment boundary in a way that the straight shape of the boundary is maintained. An important question for the future is how the signaling pathways that maintain the straight shape of compartment boundaries control mechanical tension along these boundaries.

An RNA interference screen for genes required to shape the anteroposterior compartment boundary in Drosophila identifies the Eph receptor.

The formation of straight compartment boundaries separating groups of cells with distinct fates and functions is an evolutionarily conserved strategy during animal development. The physical mechanisms that shape compartment boundaries have recently been further elucidated, however, the molecular mechanisms that underlie compartment boundary formation and maintenance remain poorly understood. Here, we report on the outcome of an RNA interference screen aimed at identifying novel genes involved in maintaining the straight shape of the anteroposterior compartment boundary in Drosophila wing imaginal discs. Out of screening 3114 transgenic RNA interference lines targeting a total of 2863 genes, we identified a single novel candidate that interfered with the formation of a straight anteroposterior compartment boundary. Interestingly, the targeted gene encodes for the Eph receptor tyrosine kinase, an evolutionarily conserved family of signal transducers that has previously been shown to be important for maintaining straight compartment boundaries in vertebrate embryos. Our results identify a hitherto unknown role of the Eph receptor tyrosine kinase in Drosophila and suggest that Eph receptors have important functions in shaping compartment boundaries in both vertebrate and insect development.

Local increases in mechanical tension shape compartment boundaries by biasing cell intercalations.

Mechanical forces play important roles during tissue organization in developing animals. Many tissues are organized into adjacent, nonmixing groups of cells termed compartments. Boundaries between compartments display a straight morphology and are associated with signaling centers that are important for tissue growth and patterning. Local increases in mechanical tension at cell junctions along compartment boundaries have recently been shown to prevent cell mixing and to maintain straight boundaries. The cellular mechanisms by which local increases in mechanical tension prevent cell mixing at compartment boundaries, however, remain poorly understood. Here, we have used live imaging and quantitative image analysis to determine cellular dynamics at and near the anteroposterior compartment boundaries of the Drosophila pupal abdominal epidermis. We show that cell mixing within compartments involves multiple cell intercalations. Frequency and orientation of cell intercalations are unchanged along the compartment boundaries; rather, an asymmetry in the shrinkage of junctions during intercalation is biased, resulting in cell rearrangements that suppress cell mixing. Simulations of tissue growth show that local increases in mechanical tension can account for this bias in junctional shrinkage. We conclude that local increases in mechanical tension maintain cell populations separate by influencing junctional rearrangements during cell intercalation.