Mechanical stress combines with planar polarised patterning during metaphase to orient embryonic epithelial cell divisions.

The planar orientation of cell division (OCD) is important for epithelial morphogenesis and homeostasis. Here, we ask how mechanics and antero-posterior (AP) patterning combine to influence the first divisions after gastrulation in the Drosophila embryonic epithelium. We analyse hundreds of cell divisions and show that stress anisotropy, notably from compressive forces, can reorient division directly in metaphase. Stress anisotropy influences the OCD by imposing metaphase cell elongation, despite mitotic rounding, and overrides interphase cell elongation. In strongly elongated cells, the mitotic spindle adapts its length to, and hence its orientation is constrained by, the cell long axis. Alongside mechanical cues, we find a tissue-wide bias of the mitotic spindle orientation towards AP-patterned planar polarised Myosin-II. This spindle bias is lost in an AP-patterning mutant. Thus, a patterning-induced mitotic spindle orientation bias overrides mechanical cues in mildly elongated cells, whereas in strongly elongated cells the spindle is constrained close to the high stress axis.

Polarised cell intercalation during Drosophila axis extension is robust to an orthogonal pull by the invaginating mesoderm.

As tissues grow and change shape during animal development, they physically pull and push on each other, and these mechanical interactions can be important for morphogenesis. During Drosophila gastrulation, mesoderm invagination temporally overlaps with the convergence and extension of the ectodermal germband; the latter is caused primarily by Myosin II-driven polarised cell intercalation. Here, we investigate the impact of mesoderm invagination on ectoderm extension, examining possible mechanical and mechanotransductive effects on Myosin II recruitment and polarised cell intercalation. We find that the germband ectoderm is deformed by the mesoderm pulling in the orthogonal direction to germband extension (GBE), showing mechanical coupling between these tissues. However, we do not find a significant change in Myosin II planar polarisation in response to mesoderm invagination, nor in the rate of junction shrinkage leading to neighbour exchange events. We conclude that the main cellular mechanism of axis extension, polarised cell intercalation, is robust to the mesoderm invagination pull. We find, however, that mesoderm invagination slows down the rate of anterior-posterior cell elongation that contributes to axis extension, counteracting the tension from the endoderm invagination, which pulls along the direction of GBE.

Different temporal requirements for tartan and wingless in the formation of contractile interfaces at compartmental boundaries.

Compartmental boundaries physically separate developing tissues into distinct regions, which is fundamental for the organisation of the body plan in both insects and vertebrates. In many examples, this physical segregation is caused by a regulated increase in contractility of the actomyosin cortex at boundary cell-cell interfaces, a property important in developmental morphogenesis beyond compartmental boundary formation. We performed an unbiased screening approach to identify cell surface receptors required for actomyosin enrichment and polarisation at parasegmental boundaries (PSBs) in early Drosophila embryos, from the start of germband extension at gastrulation and throughout the germband extended stages (stages 6 to 11). First, we find that Tartan is required during germband extension for actomyosin enrichment at PSBs, confirming an earlier report. Next, by following in real time the dynamics of loss of boundary straightness in tartan mutant embryos compared with wild-type and ftz mutant embryos, we show that Tartan is required during germband extension but not beyond. We identify candidate genes that could take over from Tartan at PSBs and confirm that at germband extended stages, actomyosin enrichment at PSBs requires Wingless signalling.

Adhesion-regulated junction slippage controls cell intercalation dynamics in an Apposed-Cortex Adhesion Model.

Cell intercalation is a key cell behaviour of morphogenesis and wound healing, where local cell neighbour exchanges can cause dramatic tissue deformations such as body axis extension. Substantial experimental work has identified the key molecular players facilitating intercalation, but there remains a lack of consensus and understanding of their physical roles. Existing biophysical models that represent cell-cell contacts with single edges cannot study cell neighbour exchange as a continuous process, where neighbouring cell cortices must uncouple. Here, we develop an Apposed-Cortex Adhesion Model (ACAM) to understand active cell intercalation behaviours in the context of a 2D epithelial tissue. The junctional actomyosin cortex of every cell is modelled as a continuous viscoelastic rope-loop, explicitly representing cortices facing each other at bicellular junctions and the adhesion molecules that couple them. The model parameters relate directly to the properties of the key subcellular players that drive dynamics, providing a multi-scale understanding of cell behaviours. We show that active cell neighbour exchanges can be driven by purely junctional mechanisms. Active contractility and cortical turnover in a single bicellular junction are sufficient to shrink and remove a junction. Next, a new, orthogonal junction extends passively. The ACAM reveals how the turnover of adhesion molecules regulates tension transmission and junction deformation rates by controlling slippage between apposed cell cortices. The model additionally predicts that rosettes, which form when a vertex becomes common to many cells, are more likely to occur in actively intercalating tissues with strong friction from adhesion molecules.

Cell sorting and morphogenesis in early Drosophila embryos.

The regionalisation of growing tissues into compartments that do not mix is thought to be a common motif of animal development. Compartments and compartmental boundaries were discovered by lineage studies in the model organism Drosophila. Since then, many compartment boundaries have been identified in developing tissues, from insects to vertebrates. These are important for animal development, because boundaries localize signalling centres that control tissue morphogenesis. Compartment boundaries are boundaries of lineage restriction, where specific mechanisms keep boundaries straight and cells segregated. Here, we review the mechanisms of cell sorting at boundaries found in early Drosophila embryos. The parasegmental boundaries, separating anterior from posterior compartments in the embryo, keep cells segregated by increasing actomyosin contractility at boundary cell-cell interfaces. Differential actomyosin contractility in turn promotes fold formation and orients cell division. Earlier in development, actomyosin differentials are also important for cell sorting during axis extension. Specific cell surface asymmetries and signalling pathways are required to initiate and maintain these actomyosin differentials.

The tricellular vertex-specific adhesion molecule Sidekick facilitates polarised cell intercalation during Drosophila axis extension.

In epithelia, tricellular vertices are emerging as important sites for the regulation of epithelial integrity and function. Compared to bicellular contacts, however, much less is known. In particular, resident proteins at tricellular vertices were identified only at occluding junctions, with none known at adherens junctions (AJs). In a previous study, we discovered that in Drosophila embryos, the adhesion molecule Sidekick (Sdk), well-known in invertebrates and vertebrates for its role in the visual system, localises at tricellular vertices at the level of AJs. Here, we survey a wide range of Drosophila epithelia and establish that Sdk is a resident protein at tricellular AJs (tAJs), the first of its kind. Clonal analysis showed that two cells, rather than three cells, contributing Sdk are sufficient for tAJ localisation. Super-resolution imaging using structured illumination reveals that Sdk proteins form string-like structures at vertices. Postulating that Sdk may have a role in epithelia where AJs are actively remodelled, we analysed the phenotype of sdk null mutant embryos during Drosophila axis extension using quantitative methods. We find that apical cell shapes are abnormal in sdk mutants, suggesting a defect in tissue remodelling during convergence and extension. Moreover, adhesion at apical vertices is compromised in rearranging cells, with apical tears in the cortex forming and persisting throughout axis extension, especially at the centres of rosettes. Finally, we show that polarised cell intercalation is decreased in sdk mutants. Mathematical modelling of the cell behaviours supports the notion that the T1 transitions of polarised cell intercalation are delayed in sdk mutants, in particular in rosettes. We propose that this delay, in combination with a change in the mechanical properties of the converging and extending tissue, causes the abnormal apical cell shapes in sdk mutant embryos.

Suppression of epithelial folding at actomyosin-enriched compartment boundaries downstream of Wingless signalling in Drosophila.

Epithelial folding shapes embryos and tissues during development. Here, we investigate the coupling between epithelial folding and actomyosin-enriched compartmental boundaries. The mechanistic relationship between the two is unclear, because actomyosin-enriched boundaries are not necessarily associated with folds. Also, some cases of epithelial folding occur independently of actomyosin contractility. We investigated the shallow folds called parasegment grooves that form at boundaries between anterior and posterior compartments in the early Drosophila embryo. We demonstrate that formation of these folds requires the presence of an actomyosin enrichment along the boundary cell-cell contacts. These enrichments, which require Wingless signalling, increase interfacial tension not only at the level of the adherens junctions but also along the lateral surfaces. We find that epithelial folding is normally under inhibitory control because different genetic manipulations, including depletion of the Myosin II phosphatase Flapwing, increase the depth of folds at boundaries. Fold depth correlates with the levels of Bazooka (Baz), the Par-3 homologue, along the boundary cell-cell contacts. Moreover, Wingless and Hedgehog signalling have opposite effects on fold depth at the boundary that correlate with changes in Baz planar polarity.

Mechanical Coupling between Endoderm Invagination and Axis Extension in Drosophila.

How genetic programs generate cell-intrinsic forces to shape embryos is actively studied, but less so how tissue-scale physical forces impact morphogenesis. Here we address the role of the latter during axis extension, using Drosophila germband extension (GBE) as a model. We found previously that cells elongate in the anteroposterior (AP) axis in the extending germband, suggesting that an extrinsic tensile force contributed to body axis extension. Here we further characterized the AP cell elongation patterns during GBE, by tracking cells and quantifying their apical cell deformation over time. AP cell elongation forms a gradient culminating at the posterior of the embryo, consistent with an AP-oriented tensile force propagating from there. To identify the morphogenetic movements that could be the source of this extrinsic force, we mapped gastrulation movements temporally using light sheet microscopy to image whole Drosophila embryos. We found that both mesoderm and endoderm invaginations are synchronous with the onset of GBE. The AP cell elongation gradient remains when mesoderm invagination is blocked but is abolished in the absence of endoderm invagination. This suggested that endoderm invagination is the source of the tensile force. We next looked for evidence of this force in a simplified system without polarized cell intercalation, in acellular embryos. Using Particle Image Velocimetry, we identify posteriorwards Myosin II flows towards the presumptive posterior endoderm, which still undergoes apical constriction in acellular embryos as in wildtype. We probed this posterior region using laser ablation and showed that tension is increased in the AP orientation, compared to dorsoventral orientation or to either orientations more anteriorly in the embryo. We propose that apical constriction leading to endoderm invagination is the source of the extrinsic force contributing to germband extension. This highlights the importance of physical interactions between tissues during morphogenesis.

Geometry can provide long-range mechanical guidance for embryogenesis.

Downstream of gene expression, effectors such as the actomyosin contractile machinery drive embryo morphogenesis. During Drosophila embryonic axis extension, actomyosin has a specific planar-polarised organisation, which is responsible for oriented cell intercalation. In addition to these cell rearrangements, cell shape changes also contribute to tissue deformation. While cell-autonomous dynamics are well described, understanding the tissue-scale behaviour challenges us to solve the corresponding mechanical problem at the scale of the whole embryo, since mechanical resistance of all neighbouring epithelia will feedback on individual cells. Here we propose a novel numerical approach to compute the whole-embryo dynamics of the actomyosin-rich apical epithelial surface. We input in the model specific patterns of actomyosin contractility, such as the planar-polarisation of actomyosin in defined ventro-lateral regions of the embryo. Tissue strain rates and displacements are then predicted over the whole embryo surface according to the global balance of stresses and the material behaviour of the epithelium. Epithelia are modelled using a rheological law that relates the rate of deformation to the local stresses and actomyosin anisotropic contractility. Predicted flow patterns are consistent with the cell flows observed when imaging Drosophila axis extension in toto, using light sheet microscopy. The agreement between model and experimental data indicates that the anisotropic contractility of planar-polarised actomyosin in the ventro-lateral germband tissue can directly cause the tissue-scale deformations of the whole embryo. The three-dimensional mechanical balance is dependent on the geometry of the embryo, whose curved surface is taken into account in the simulations. Importantly, we find that to reproduce experimental flows, the model requires the presence of the cephalic furrow, a fold located anteriorly of the extending tissues. The presence of this geometric feature, through the global mechanical balance, guides the flow and orients extension towards the posterior end.

Subcellular localisations of the CPTI collection of YFP-tagged proteins in Drosophila embryos.

A key challenge in the post-genomic area is to identify the function of the genes discovered, with many still uncharacterised in all metazoans. A first step is transcription pattern characterisation, for which we now have near whole-genome coverage in Drosophila. However, we have much more limited information about the expression and subcellular localisation of the corresponding proteins. The Cambridge Protein Trap Consortium generated, via piggyBac transposition, over 600 novel YFP-trap proteins tagging just under 400 Drosophila loci. Here, we characterise the subcellular localisations and expression patterns of these insertions, called the CPTI lines, in Drosophila embryos. We have systematically analysed subcellular localisations at cellularisation (stage 5) and recorded expression patterns at stage 5, at mid-embryogenesis (stage 11) and at late embryogenesis (stages 15-17). At stage 5, 31% of the nuclear lines (41) and 26% of the cytoplasmic lines (67) show discrete localisations that provide clues on the function of the protein and markers for organelles or regions, including nucleoli, the nuclear envelope, nuclear speckles, centrosomes, mitochondria, the endoplasmic reticulum, Golgi, lysosomes and peroxisomes. We characterised the membranous/cortical lines (102) throughout stage 5 to 10 during epithelial morphogenesis, documenting their apico-basal position and identifying those secreted in the extracellular space. We identified the tricellular vertices as a specialized membrane domain marked by the integral membrane protein Sidekick. Finally, we categorised the localisation of the membranous/cortical proteins during cytokinesis.

Analysis of the expression patterns, subcellular localisations and interaction partners of Drosophila proteins using a pigP protein trap library.

Although we now have a wealth of information on the transcription patterns of all the genes in the Drosophila genome, much less is known about the properties of the encoded proteins. To provide information on the expression patterns and subcellular localisations of many proteins in parallel, we have performed a large-scale protein trap screen using a hybrid piggyBac vector carrying an artificial exon encoding yellow fluorescent protein (YFP) and protein affinity tags. From screening 41 million embryos, we recovered 616 verified independent YFP-positive lines representing protein traps in 374 genes, two-thirds of which had not been tagged in previous P element protein trap screens. Over 20 different research groups then characterized the expression patterns of the tagged proteins in a variety of tissues and at several developmental stages. In parallel, we purified many of the tagged proteins from embryos using the affinity tags and identified co-purifying proteins by mass spectrometry. The fly stocks are publicly available through the Kyoto Drosophila Genetics Resource Center. All our data are available via an open access database (Flannotator), which provides comprehensive information on the expression patterns, subcellular localisations and in vivo interaction partners of the trapped proteins. Our resource substantially increases the number of available protein traps in Drosophila and identifies new markers for cellular organelles and structures.

Tissue tectonics: morphogenetic strain rates, cell shape change and intercalation.

The dynamic reshaping of tissues during morphogenesis results from a combination of individual cell behaviors and collective cell rearrangements. However, a comprehensive framework to unambiguously measure and link cell behavior to tissue morphogenesis is lacking. Here we introduce such a kinematic framework, bridging cell and tissue behaviors at an intermediate, mesoscopic, level of cell clusters or domains. By measuring domain deformation in terms of the relative motion of cell positions and the evolution of their shapes, we characterized the basic invariant quantities that measure fundamental classes of cell behavior, namely tensorial rates of cell shape change and cell intercalation. In doing so we introduce an explicit definition of cell intercalation as a continuous process. We mapped strain rates spatiotemporally in three models of tissue morphogenesis, gaining insight into morphogenetic mechanisms. Our quantitative approach has broad relevance for the precise characterization and comparison of morphogenetic phenotypes.

Establishment and maintenance of compartmental boundaries: role of contractile actomyosin barriers.

During animal development, tissues and organs are partitioned into compartments that do not intermix. This organizing principle is essential for correct tissue morphogenesis. Given that cell sorting defects during compartmentalization in humans are thought to cause malignant invasion and congenital defects such as cranio-fronto-nasal syndrome, identifying the molecular and cellular mechanisms that keep cells apart at boundaries between compartments is important. In both vertebrates and invertebrates, transcription factors and short-range signalling pathways, such as EPH/Ephrin, Hedgehog, or Notch signalling, govern compartmental cell sorting. However, the mechanisms that mediate cell sorting downstream of these factors have remained elusive for decades. Here, we review recent data gathered in Drosophila that suggest that the generation of cortical tensile forces at compartmental boundaries by the actomyosin cytoskeleton could be a general mechanism that inhibits cell mixing between compartments.

Embryo-scale epithelial buckling forms a propagating furrow that initiates gastrulation.

Cell apical constriction driven by actomyosin contraction forces is a conserved mechanism during tissue folding in embryo development. While much is now understood of the molecular mechanism responsible for apical constriction and of the tissue-scale integration of the ensuing in-plane deformations, it is still not clear if apical actomyosin contraction forces are necessary or sufficient per se to drive tissue folding. To tackle this question, we use the Drosophila embryo model system that forms a furrow on the ventral side, initiating mesoderm internalization. Past computational models support the idea that cell apical contraction forces may not be sufficient and that active or passive cell apico-basal forces may be necessary to drive cell wedging leading to tissue furrowing. By using 3D computational modelling and in toto embryo image analysis and manipulation, we now challenge this idea and show that embryo-scale force balance at the tissue surface, rather than cell-autonomous shape changes, is necessary and sufficient to drive a buckling of the epithelial surface forming a furrow which propagates and initiates embryo gastrulation.

Actomyosin-Driven Tension at Compartmental Boundaries Orients Cell Division Independently of Cell Geometry In Vivo.

Cell shape is known to influence the plane of cell division. In vitro, mechanical constraints can also orient mitoses; however, in vivo it is not clear whether tension can orient the mitotic spindle directly, because tissue-scale forces can change cell shape. During segmentation of the Drosophila embryo, actomyosin is enriched along compartment boundaries forming supracellular cables that keep cells segregated into distinct compartments. Here, we show that these actomyosin cables orient the planar division of boundary cells perpendicular to the boundaries. This bias overrides the influence of cell shape, when cells are mildly elongated. By decreasing actomyosin cable tension with laser ablation or, conversely, ectopically increasing tension with laser wounding, we demonstrate that local tension is necessary and sufficient to orient mitoses in vivo. This involves capture of the spindle pole by the actomyosin cortex. These findings highlight the importance of actomyosin-mediated tension in spindle orientation in vivo.

A screen for genes regulating the wingless gradient in Drosophila embryos.

During the development of the Drosophila embryonic epidermis, the secreted Wingless protein initially spreads symmetrically from its source. At later stages, Wingless becomes asymmetrically distributed in a Hedgehog-dependent manner, to control the patterning of the embryonic epidermis. When Wingless is misexpressed in engrailed cells in hedgehog heterozygous mutant embryos, larvae show a dominant phenotype consisting of patches of naked cuticle in denticle belts. This dose-sensitive phenotype is a direct consequence of a change in Wg protein distribution. We used this phenotype to carry out a screen for identifying genes regulating Wingless distribution or transport in the embryonic epidermis. Using a third chromosome deficiency collection, we found several genomic regions that showed a dominant interaction. After using a secondary screen to test for mutants and smaller deficiencies, we identified three interacting genes: dally, notum, and brahma. We confirmed that dally, as well as its homolog dally-like, and notum affect Wingless distribution in the embryonic epidermis, directly or indirectly. Thus, our assay can be used effectively to screen for genes regulating Wingless distribution or transport.

Performing Chromophore-Assisted Laser Inactivation in Drosophila Embryos Using GFP.

Chromophore-assisted laser inactivation (CALI) is an optogenetic technique in which light-induced release of reactive oxygen species triggers acute inactivation of a protein of interest, with high spatial and temporal resolution. At its simplest, selective protein inactivation can be achieved via the genetic fusion of the protein to a photosensitizer such as EGFP, and using standard optical setups such as laser scanning confocal microscopes. Although use of CALI in Drosophila is relatively recent, this technique can be a powerful complement to developmental genetics, especially in vivo as it allows visualization of the immediate consequences of local protein inactivation when coupled to time-lapse microscopy analysis. In addition to providing examples of protocols, this chapter is intended as a conceptual framework to support the rational design of CALI experiments.

Unipolar distributions of junctional Myosin II identify cell stripe boundaries that drive cell intercalation throughout Drosophila axis extension.

Convergence and extension movements elongate tissues during development. Drosophila germ-band extension (GBE) is one example, which requires active cell rearrangements driven by Myosin II planar polarisation. Here, we develop novel computational methods to analyse the spatiotemporal dynamics of Myosin II during GBE, at the scale of the tissue. We show that initial Myosin II bipolar cell polarization gives way to unipolar enrichment at parasegmental boundaries and two further boundaries within each parasegment, concomitant with a doubling of cell number as the tissue elongates. These boundaries are the primary sites of cell intercalation, behaving as mechanical barriers and providing a mechanism for how cells remain ordered during GBE. Enrichment at parasegment boundaries during GBE is independent of Wingless signaling, suggesting pair-rule gene control. Our results are consistent with recent work showing that a combinatorial code of Toll-like receptors downstream of pair-rule genes contributes to Myosin II polarization via local cell-cell interactions. We propose an updated cell-cell interaction model for Myosin II polarization that we tested in a vertex-based simulation.

Tension and epithelial morphogenesis in Drosophila early embryos.

During morphogenesis, tissues are shaped by cell behaviors such as apical cell constriction and cell intercalation, which are the result of cell intrinsic forces, but are also shaped passively by forces acting on the cells. The latter extrinsic forces can be produced either within the deforming tissue by the tissue-scale integration of intrinsic forces, or outside the tissue by other tissue movements or by fluid flows. Here we review the intrinsic and extrinsic forces that sculpt the epithelium of early Drosophila embryos, focusing on three conserved morphogenetic processes: tissue internalization, axis extension, and segment boundary formation. Finally, we look at how the actomyosin cytoskeleton forms force-generating structures that power these three morphogenetic events at the cell and the tissue scales.

Epithelial polarity and morphogenesis.

The adult form of a multicellular organism is shaped by a series of morphogenetic processes that organise the body into tissues and organs. Most of these events involve the deformation of sheets of epithelial cells that are highly polarised along their apical-basal axes and attached to each other by lateral junctions. Here we discuss the role played by modifications in the apical-basal polarity system in driving morphogenesis, with an emphasis on well-characterised events during Drosophila development. Changing the activity of polarity factors can alter the relative sizes of the apical, lateral and basal domains. This can drive transitions between cuboidal, columnar and squamous epithelial morphologies, to increase or decrease the surface area of an epithelial sheet. These changes can also cause epithelial cells to become wedge-shaped, which can drive tissue bending and invagination. In addition, it has recently emerged that the activity of apical-basal polarity factors can also be modulated in a planar polarised manner. By affecting the contractility of the actomyosin cytoskeleton and the stability of adherens junctions, changes within the plane of the epithelium can cause cell rearrangements that contribute to convergence and extension movements, boundary formation and cell alignment.