Cellular defects resulting from disease-related myosin II mutations in Drosophila.

The nonmuscle myosin II motor protein produces forces that are essential to driving the cell movements and cell shape changes that generate tissue structure. Mutations in myosin II that are associated with human diseases are predicted to disrupt critical aspects of myosin function, but the mechanisms that translate altered myosin activity into specific changes in tissue organization and physiology are not well understood. Here we use the Drosophila embryo to model human disease mutations that affect myosin motor activity. Using in vivo imaging and biophysical analysis, we show that engineering human MYH9-related disease mutations into Drosophila myosin II produces motors with altered organization and dynamics that fail to drive rapid cell movements, resulting in defects in epithelial morphogenesis. In embryos that express the Drosophila myosin motor variants R707C or N98K and have reduced levels of wild-type myosin, myosin motors are correctly planar polarized and generate anisotropic contractile tension in the tissue. However, expression of these motor variants is associated with a cellular-scale reduction in the speed of cell intercalation, resulting in a failure to promote full elongation of the body axis. In addition, these myosin motor variants display slowed turnover and aberrant aggregation at the cell cortex, indicating that mutations in the motor domain influence mesoscale properties of myosin organization and dynamics. These results demonstrate that disease-associated mutations in the myosin II motor domain disrupt specific aspects of myosin localization and activity during cell intercalation, linking molecular changes in myosin activity to defects in tissue morphogenesis.

SEGGA: a toolset for rapid automated analysis of epithelial cell polarity and dynamics.

Epithelial remodeling determines the structure of many organs in the body through changes in cell shape, polarity and behavior and is a major area of study in developmental biology. Accurate and high-throughput methods are necessary to systematically analyze epithelial organization and dynamics at single-cell resolution. We developed SEGGA, an easy-to-use software for automated image segmentation, cell tracking and quantitative analysis of cell shape, polarity and behavior in epithelial tissues. SEGGA is free, open source, and provides a full suite of tools that allow users with no prior computational expertise to independently perform all steps of automated image segmentation, semi-automated user-guided error correction, and data analysis. Here we use SEGGA to analyze changes in cell shape, cell interactions and planar polarity during convergent extension in the Drosophila embryo. These studies demonstrate that planar polarity is rapidly established in a spatiotemporally regulated pattern that is dynamically remodeled in response to changes in cell orientation. These findings reveal an unexpected plasticity that maintains coordinated planar polarity in actively moving populations through the continual realignment of cell polarity with the tissue axes.

A positional Toll receptor code directs convergent extension in Drosophila.

Elongation of the head-to-tail body axis by convergent extension is a conserved developmental process throughout metazoans. In Drosophila, patterns of transcription factor expression provide spatial cues that induce systematically oriented cell movements and promote tissue elongation. However, the mechanisms by which patterned transcriptional inputs control cell polarity and behaviour have long been elusive. We demonstrate that three Toll family receptors, Toll-2, Toll-6 and Toll-8, are expressed in overlapping transverse stripes along the anterior-posterior axis and act in combination to direct planar polarity and polarized cell rearrangements during convergent extension. Simultaneous disruption of all three receptors strongly reduces actomyosin-driven junctional remodelling and axis elongation, and an ectopic stripe of Toll receptor expression is sufficient to induce planar polarized actomyosin contractility. These results demonstrate that tissue-level patterns of Toll receptor expression provide spatial signals that link positional information from the anterior-posterior patterning system to the essential cell behaviours that drive convergent extension.

Spatiotemporal control of epithelial remodeling by regulated myosin phosphorylation.

Spatiotemporally regulated actomyosin contractility generates the forces that drive epithelial cell rearrangements and tissue remodeling. Phosphorylation of the myosin II regulatory light chain (RLC) promotes the assembly of myosin monomers into active contractile filaments and is an essential mechanism regulating the level of myosin activity. However, the effects of phosphorylation on myosin localization, dynamics, and function during epithelial remodeling are not well understood. In Drosophila, planar polarized myosin contractility is required for oriented cell rearrangements during elongation of the body axis. We show that regulated myosin phosphorylation influences spatial and temporal properties of contractile behavior at molecular, cellular, and tissue length scales. Expression of myosin RLC variants that prevent or mimic phosphorylation both disrupt axis elongation, but have distinct effects at the molecular and cellular levels. Unphosphorylatable RLC produces fewer, slower cell rearrangements, whereas phosphomimetic RLC accelerates rearrangement and promotes higher-order cell interactions. Quantitative live imaging and biophysical approaches reveal that both phosphovariants reduce myosin planar polarity and mechanical anisotropy, altering the orientation of cell rearrangements during axis elongation. Moreover, the localized myosin activator Rho-kinase is required for spatially regulated myosin activity, even when the requirement for phosphorylation is bypassed by the expression of phosphomimetic myosin RLC. These results indicate that myosin phosphorylation influences both the level and the spatiotemporal regulation of myosin activity, linking molecular properties of myosin activity to tissue morphogenesis.

Regulation of cytoskeletal organization and junctional remodeling by the atypical cadherin Fat.

The atypical cadherin Fat is a conserved regulator of planar cell polarity, but the mechanisms by which Fat controls cell shape and tissue structure are not well understood. Here, we show that Fat is required for the planar polarized organization of actin denticle precursors, adherens junction proteins and microtubules in the epidermis of the late Drosophila embryo. In wild-type embryos, spatially regulated cell-shape changes and rearrangements organize cells into highly aligned columns. Junctional remodeling is suppressed at dorsal and ventral cell boundaries, where adherens junction proteins accumulate. By contrast, adherens junction proteins fail to accumulate to the wild-type extent and all cell boundaries are equally engaged in junctional remodeling in fat mutants. The effects of loss of Fat on cell shape and junctional localization, but not its role in denticle organization, are recapitulated by mutations in Expanded, an upstream regulator of the conserved Hippo pathway, and mutations in Hippo and Warts, two kinases in the Hippo kinase cascade. However, the cell shape and planar polarity defects in fat mutants are not suppressed by removing the transcriptional co-activator Yorkie, suggesting that these roles of Fat are independent of Yorkie-mediated transcription. The effects of Fat on cell shape, junctional remodeling and microtubule localization are recapitulated by expression of activated Notch. These results demonstrate that cell shape, junctional localization and cytoskeletal planar polarity in the Drosophila embryo are regulated by a common signal provided by the atypical cadherin Fat and suggest that Fat influences tissue organization through its role in polarized junctional remodeling.

Elaborating polarity: PAR proteins and the cytoskeleton.

Cell polarity is essential for cells to divide asymmetrically, form spatially restricted subcellular structures and participate in three-dimensional multicellular organization. PAR proteins are conserved polarity regulators that function by generating cortical landmarks that establish dynamic asymmetries in the distribution of effector proteins. Here, we review recent findings on the role of PAR proteins in cell polarity in C. elegans and Drosophila, and emphasize the links that exist between PAR networks and cytoskeletal proteins that both regulate PAR protein localization and act as downstream effectors to elaborate polarity within the cell.

A single-cell atlas of spatial and temporal gene expression in the mouse cranial neural plate.

The formation of the mammalian brain requires regionalization and morphogenesis of the cranial neural plate, which transforms from an epithelial sheet into a closed tube that provides the structural foundation for neural patterning and circuit formation. Sonic hedgehog (SHH) signaling is important for cranial neural plate patterning and closure, but the transcriptional changes that give rise to the spatially regulated cell fates and behaviors that build the cranial neural tube have not been systematically analyzed. Here we used single-cell RNA sequencing to generate an atlas of gene expression at six consecutive stages of cranial neural tube closure in the mouse embryo. Ordering transcriptional profiles relative to the major axes of gene expression predicted spatially regulated expression of 870 genes along the anterior-posterior and mediolateral axes of the cranial neural plate and reproduced known expression patterns with over 85% accuracy. Single-cell RNA sequencing of embryos with activated SHH signaling revealed distinct SHH-regulated transcriptional programs in the developing forebrain, midbrain, and hindbrain, suggesting a complex interplay between anterior-posterior and mediolateral patterning systems. These results define a spatiotemporally resolved map of gene expression during cranial neural tube closure and provide a resource for investigating the transcriptional events that drive early mammalian brain development.

Translating cell polarity into tissue elongation.

Planar cell polarity, the orientation of single-cell asymmetries within the plane of a multicellular tissue, is essential to generating the shape and dimensions of organs and organisms. Planar polarity systems align cell behavior with the body axes and orient the cellular processes that lead to tissue elongation. Using Drosophila as a model system, significant progress has been made toward understanding how planar polarity is generated by biochemical and mechanical signals. Recent studies using time-lapse imaging reveal that cells engage in a number of active behaviors whose orientation and dynamics translate planar cell polarity into tissue elongation. Here we review recent progress in understanding the cellular mechanisms that link planar polarity to large-scale changes in tissue structure.

Multicellular rosettes link mesenchymal-epithelial transition to radial intercalation in the mouse axial mesoderm.

Mesenchymal-epithelial transitions are fundamental drivers of development and disease, but how these behaviors generate epithelial structure is not well understood. Here, we show that mesenchymal-epithelial transitions promote epithelial organization in the mouse node and notochordal plate through the assembly and radial intercalation of three-dimensional rosettes. Axial mesoderm rosettes acquire junctional and apical polarity, develop a central lumen, and dynamically expand, coalesce, and radially intercalate into the surface epithelium, converting mesenchymal-epithelial transitions into higher-order tissue structure. In mouse Par3 mutants, axial mesoderm rosettes establish central tight junction polarity but fail to form an expanded apical domain and lumen. These defects are associated with altered rosette dynamics, delayed radial intercalation, and formation of a small, fragmented surface epithelial structure. These results demonstrate that three-dimensional rosette behaviors translate mesenchymal-epithelial transitions into collective radial intercalation and epithelial formation, providing a strategy for building epithelial sheets from individual self-organizing units in the mammalian embryo.

Epithelial organization: may the force be with you.

Multicellular organization is determined by the balance of forces between cells as much as by the expression of genes. A recent study in Drosophila combines physical modeling with experimental measurements and mechanical perturbations to shed light on the processes that influence cell patterns in vivo.

Multicellular rosette formation links planar cell polarity to tissue morphogenesis.

Elongation of the body axis is accompanied by the assembly of a polarized cytoarchitecture that provides the basis for directional cell behavior. We find that planar polarity in the Drosophila embryo is established through a sequential enrichment of actin-myosin cables and adherens junction proteins in complementary surface domains. F-actin accumulation at AP interfaces represents the first break in planar symmetry and occurs independently of proper junctional protein distribution at DV interfaces. Polarized cells engage in a novel program of locally coordinated behavior to generate multicellular rosette structures that form and resolve in a directional fashion. Actin-myosin structures align across multiple cells during rosette formation, and adherens junction proteins assemble in a stepwise fashion during rosette resolution. Patterning genes essential for axis elongation selectively affect the frequency and directionality of rosette formation. We propose that the generation of higher-order rosette structures links local cell interactions to global tissue reorganization during morphogenesis.

Oscillatory behaviors and hierarchical assembly of contractile structures in intercalating cells.

Fluctuations in the size of the apical cell surface have been associated with apical constriction and tissue invagination. However, it is currently not known if apical oscillatory behaviors are a unique property of constricting cells or if they constitute a universal feature of the force balance between cells in multicellular tissues. Here, we set out to determine whether oscillatory cell behaviors occur in parallel with cell intercalation during the morphogenetic process of axis elongation in the Drosophila embryo. We applied multi-color, time-lapse imaging of living embryos and SIESTA, an integrated tool for automated and semi-automated cell segmentation, tracking, and analysis of image sequences. Using SIESTA, we identified cycles of contraction and expansion of the apical surface in intercalating cells and characterized them at the molecular, cellular, and tissue scales. We demonstrate that apical oscillations are anisotropic, and this anisotropy depends on the presence of intact cell-cell junctions and spatial cues provided by the anterior-posterior patterning system. Oscillatory cell behaviors during axis elongation are associated with the hierarchical assembly and disassembly of contractile actomyosin structures at the medial cortex of the cell, with actin localization preceding myosin II and with the localization of both proteins preceding changes in cell shape. We discuss models to explain how the architecture of cytoskeletal networks regulates their contractile behavior and the mechanisms that give rise to oscillatory cell behaviors in intercalating cells.

Toll receptors remodel epithelia by directing planar-polarized Src and PI3K activity.

Toll-like receptors are essential for animal development and survival, with conserved roles in innate immunity, tissue patterning, and cell behavior. The mechanisms by which Toll receptors signal to the nucleus are well characterized, but how Toll receptors generate rapid, localized signals at the cell membrane to produce acute changes in cell polarity and behavior is not known. We show that Drosophila Toll receptors direct epithelial convergent extension by inducing planar-polarized patterns of Src and PI3-kinase (PI3K) activity. Toll receptors target Src activity to specific sites at the membrane, and Src recruits PI3K to the Toll-2 complex through tyrosine phosphorylation of the Toll-2 cytoplasmic domain. Reducing Src or PI3K activity disrupts planar-polarized myosin assembly, cell intercalation, and convergent extension, whereas constitutive Src activity promotes ectopic PI3K and myosin cortical localization. These results demonstrate that Toll receptors direct cell polarity and behavior by locally mobilizing Src and PI3K activity.

Cellular, molecular, and biophysical control of epithelial cell intercalation.

Convergent extension is a conserved mechanism for elongating tissues. In the Drosophila embryo, convergent extension is driven by planar polarized cell intercalation and is a paradigm for understanding the cellular, molecular, and biophysical mechanisms that establish tissue structure. Studies of convergent extension in Drosophila have provided key insights into the force-generating molecules that promote convergent extension in epithelial tissues, as well as the global systems of spatial information that systematically organize these cell behaviors. A general framework has emerged in which asymmetrically localized proteins involved in cytoskeletal tension and cell adhesion direct oriented cell movements, and spatial signals provided by the Toll, Tartan, and Teneurin receptor families break planar symmetry to establish and coordinate planar cell polarity throughout the tissue. In this chapter, we describe the cellular, molecular, and biophysical mechanisms that regulate cell intercalation in the Drosophila embryo, and discuss how research in this system has revealed conserved biological principles that control the organization of multicellular tissues and animal body plans.

Abl and Canoe/Afadin mediate mechanotransduction at tricellular junctions.

Epithelial structure is generated by the dynamic reorganization of cells in response to mechanical forces. Adherens junctions transmit forces between cells, but how cells sense and respond to these forces in vivo is not well understood. We identify a mechanotransduction pathway involving the Abl tyrosine kinase and Canoe/Afadin that stabilizes cell adhesion under tension at tricellular junctions in the Drosophila embryo. Canoe is recruited to tricellular junctions in response to actomyosin contractility, and this mechanosensitivity requires Abl-dependent phosphorylation of a conserved tyrosine in the Canoe actin-binding domain. Preventing Canoe tyrosine phosphorylation destabilizes tricellular adhesion, and anchoring Canoe at tricellular junctions independently of mechanical inputs aberrantly stabilizes adhesion, arresting cell rearrangement. These results identify a force-responsive mechanism that stabilizes tricellular adhesion under tension during epithelial remodeling.

Sonic hedgehog signaling directs patterned cell remodeling during cranial neural tube closure.

Neural tube closure defects are a major cause of infant mortality, with exencephaly accounting for nearly one-third of cases. However, the mechanisms of cranial neural tube closure are not well understood. Here, we show that this process involves a tissue-wide pattern of apical constriction controlled by Sonic hedgehog (Shh) signaling. Midline cells in the mouse midbrain neuroepithelium are flat with large apical surfaces, whereas lateral cells are taller and undergo synchronous apical constriction, driving neural fold elevation. Embryos lacking the Shh effector Gli2 fail to produce appropriate midline cell architecture, whereas embryos with expanded Shh signaling, including the IFT-A complex mutants Ift122 and Ttc21b and embryos expressing activated Smoothened, display apical constriction defects in lateral cells. Disruption of lateral, but not midline, cell remodeling results in exencephaly. These results reveal a morphogenetic program of patterned apical constriction governed by Shh signaling that generates structural changes in the developing mammalian brain.