The positioning mechanics of microtubule asters in Drosophila embryo explants.

Microtubule asters are essential in localizing the action of microtubules in processes including mitosis and organelle positioning. In large cells, such as the one-cell sea urchin embryo, aster dynamics are dominated by hydrodynamic pulling forces. However, in systems with more densely positioned nuclei such as the early Drosophila embryo, which packs around 6000 nuclei within the syncytium in a crystalline-like order, it is unclear what processes dominate aster dynamics. Here, we take advantage of a cell cycle regulation Drosophila mutant to generate embryos with multiple asters, independent from nuclei. We use an ex vivo assay to further simplify this biological system to explore the forces generated by and between asters. Through live imaging, drug and optical perturbations, and theoretical modeling, we demonstrate that these asters likely generate an effective pushing force over short distances.

Long-range formation of the Bicoid gradient requires multiple dynamic modes that spatially vary across the embryo.

Morphogen gradients provide essential positional information to gene networks through their spatially heterogeneous distribution, yet how they form is still hotly contested, with multiple models proposed for different systems. Here, we focus on the transcription factor Bicoid (Bcd), a morphogen that forms an exponential gradient across the anterior-posterior (AP) axis of the early Drosophila embryo. Using fluorescence correlation spectroscopy we find there are spatial differences in Bcd diffusivity along the AP axis, with Bcd diffusing more rapidly in the posterior. We establish that such spatially varying differences in Bcd dynamics are sufficient to explain how Bcd can have a steep exponential gradient in the anterior half of the embryo and yet still have an observable fraction of Bcd near the posterior pole. In the nucleus, we demonstrate that Bcd dynamics are impacted by binding to DNA. Addition of the Bcd homeodomain to eGFP::NLS qualitatively replicates the Bcd concentration profile, suggesting this domain regulates Bcd dynamics. Our results reveal how a long-range gradient can form while retaining a steep profile through much of its range.

Curvature-Induced Cell Rearrangements in Biological Tissues.

On a curved surface, epithelial cells can adapt to geometric constraints by tilting and by exchanging their neighbors from apical to basal sides, known as an apico-basal topological transition 1 (AB-T1). The relationship between cell tilt, AB-T1s, and tissue curvature still lacks a unified understanding. Here, we propose a general framework for cell packing in curved environments and explain the formation of AB-T1s from the perspective of strain anisotropy. We find that steep curvature gradients can lead to cell tilting and induce AB-T1s. Alternatively, pressure differences across the epithelial tissue can drive AB-T1s in regions of large curvature anisotropy. The two mechanisms compete to determine the impact of tissue geometry and mechanics on optimized cell rearrangements in three dimensions.

JNK signaling in pioneer neurons organizes ventral nerve cord architecture in Drosophila embryos.

Morphogenesis of the Central Nervous System (CNS) is a complex process that obeys precise architectural rules. Yet, the mechanisms dictating these rules remain unknown. Analyzing morphogenesis of the Drosophila embryo Ventral Nerve Cord (VNC), we observe that a tight control of JNK signaling is essential for attaining the final VNC architecture. JNK signaling in a specific subset of pioneer neurons autonomously regulates the expression of Fasciclin 2 (Fas 2) and Neurexin IV (Nrx IV) adhesion molecules, probably via the transcription factor zfh1. Interfering at any step in this cascade affects fasciculation along pioneer axons, leading to secondary cumulative scaffolding defects during the structural organization of the axonal network. The global disorder of architectural landmarks ultimately influences nervous system condensation. In summary, our data point to JNK signaling in a subset of pioneer neurons as a key element underpinning VNC architecture, revealing critical milestones on the mechanism of control of its structural organization.

Colour patterns: Predicting patterns without knowing the details.

A bewildering array of colour patterns occurs across animals. New work studying five lizard species reveals that reaction-diffusion models can be remarkably predictive of future adult skin patterning, even though molecular details are unknown. This has implications for understanding how complex patterns evolve.

Slow muscles guide fast myocyte fusion to ensure robust myotome formation despite the high spatiotemporal stochasticity of fusion events.

Skeletal myogenesis is dynamic, and it involves cell-shape changes together with cell fusion and rearrangements. However, the final muscle arrangement is highly organized with striated fibers. By combining live imaging with quantitative analyses, we dissected fast-twitch myocyte fusion within the zebrafish myotome in toto. We found a strong mediolateral bias in fusion timing; however, at a cellular scale, there was heterogeneity in cell shape and the relationship between initial position of fast myocytes and resulting fusion partners. We show that the expression of the fusogen myomaker is permissive, but not instructive, in determining the spatiotemporal fusion pattern. Rather, we observed a close coordination between slow muscle rearrangements and fast myocyte fusion. In mutants that lack slow fibers, the spatiotemporal fusion pattern is substantially noisier. We propose a model in which slow muscles guide fast myocytes by funneling them close together, enhancing fusion probability. Thus, despite fusion being highly stochastic, a robust myotome structure emerges at the tissue scale.

Optogenetic control of YAP cellular localisation and function.

YAP, an effector of the Hippo signalling pathway, promotes organ growth and regeneration. Prolonged YAP activation results in uncontrolled proliferation and cancer. Therefore, exogenous regulation of YAP activity has potential translational applications. We present a versatile optogenetic construct (optoYAP) for manipulating YAP localisation, and consequently its activity and function. We attach a LOV2 domain that photocages a nuclear localisation signal (NLS) to the N-terminus of YAP. In 488 nm light, the LOV2 domain unfolds, exposing the NLS, which shuttles optoYAP into the nucleus. Nuclear import of optoYAP is reversible and tuneable by light intensity. In cell culture, activated optoYAP promotes YAP target gene expression and cell proliferation. Similarly, optofYap can be used in zebrafish embryos to modulate target genes. We demonstrate that optoYAP can override a cell's response to substrate stiffness to generate anchorage-independent growth. OptoYAP is functional in both cell culture and in vivo, providing a powerful tool to address basic research questions and therapeutic applications in regeneration and disease.

Slit-Robo signalling establishes a Sphingosine-1-phosphate gradient to polarise fin mesenchyme.

Immigration of mesenchymal cells into the growing fin and limb buds drives distal outgrowth, with subsequent tensile forces between these cells essential for fin and limb morphogenesis. Morphogens derived from the apical domain of the fin, orientate limb mesenchyme cell polarity, migration, division and adhesion. The zebrafish mutant stomp displays defects in fin morphogenesis including blister formation and associated loss of orientation and adhesion of immigrating fin mesenchyme cells. Positional cloning of stomp identifies a mutation in the gene encoding the axon guidance ligand, Slit3. We provide evidence that Slit ligands derived from immigrating mesenchyme act via Robo receptors at the apical ectodermal ridge (AER) to promote release of sphingosine-1-phosphate (S1P). S1P subsequently diffuses back to the mesenchyme to promote their polarisation, orientation, positioning and adhesion to the interstitial matrix of the fin fold. We thus demonstrate the coordination of the Slit-Robo and S1P signalling pathways in fin fold morphogenesis. Our work introduces a mechanism regulating the orientation, positioning and adhesion of its constituent cells.

DNA-damage induced cell death in yap1;wwtr1 mutant epidermal basal cells.

In a previous study, it was reported that Yap1 and Wwtr1 in zebrafish regulates the morphogenesis of the posterior body and epidermal fin fold (Kimelman et al., 2017). We report here that DNA damage induces apoptosis of epidermal basal cells (EBCs) in zebrafish yap1-/-;wwtr1-/- embryos. Specifically, these mutant EBCs exhibit active Caspase-3, Caspase-8, and γH2AX, consistent with DNA damage serving as a stimulus of the extrinsic apoptotic pathway in epidermal cells. Live imaging of zebrafish epidermal cells reveals a steady growth of basal cell size in the developing embryo, but this growth is inhibited in mutant basal cells followed by apoptosis, leading to the hypothesis that factors underscoring cell size play a role in this DNA damage-induced apoptosis phenotype. We tested two of these factors using cell stretching and substrate stiffness assays, and found that HaCaT cells cultured on stiff substrates exhibit more numerous γH2AX foci compared to ones cultured on soft substrates. Thus, our experiments suggest that substrate rigidity may modulate genomic stress in epidermal cells, and that Yap1 and Wwtr1 promotes their survival.

Condensation of the Drosophila nerve cord is oscillatory and depends on coordinated mechanical interactions.

During development, organs reach precise shapes and sizes. Organ morphology is not always obtained through growth; a classic counterexample is the condensation of the nervous system during Drosophila embryogenesis. The mechanics underlying such condensation remain poorly understood. Here, we characterize the condensation of the embryonic ventral nerve cord (VNC) at both subcellular and tissue scales. This analysis reveals that condensation is not a unidirectional continuous process but instead occurs through oscillatory contractions. The VNC mechanical properties spatially and temporally vary, and forces along its longitudinal axis are spatially heterogeneous. We demonstrate that the process of VNC condensation is dependent on the coordinated mechanical activities of neurons and glia. These outcomes are consistent with a viscoelastic model of condensation, which incorporates time delays and effective frictional interactions. In summary, we have defined the progressive mechanics driving VNC condensation, providing insights into how a highly viscous tissue can autonomously change shape and size.

Aster repulsion drives short-ranged ordering in the Drosophila syncytial blastoderm.

Biological systems are highly complex, yet notably ordered structures can emerge. During syncytial stage development of the Drosophila melanogaster embryo, nuclei synchronously divide for nine cycles within a single cell, after which most of the nuclei reach the cell cortex. The arrival of nuclei at the cortex occurs with remarkable positional order, which is important for subsequent cellularisation and morphological transformations. Yet, the mechanical principles underlying this lattice-like positional order of nuclei remain untested. Here, using quantification of nuclei position and division orientation together with embryo explants, we show that short-ranged repulsive interactions between microtubule asters ensure the regular distribution and maintenance of nuclear positions in the embryo. Such ordered nuclear positioning still occurs with the loss of actin caps and even the loss of the nuclei themselves; the asters can self-organise with similar distribution to nuclei in the wild-type embryo. The explant assay enabled us to deduce the nature of the mechanical interaction between pairs of nuclei. We used this to predict how the nuclear division axis orientation changes upon nucleus removal from the embryo cortex, which we confirmed in vivo with laser ablation. Overall, we show that short-ranged microtubule-mediated repulsive interactions between asters are important for ordering in the early Drosophila embryo and minimising positional irregularity.

Protocol for batch imaging and quantification of cellular mismatch during Drosophila embryonic heart formation.

How individual cells form precise connections with partners in a complicated environment has been a longstanding question. However, most cell matching studies have used qualitative approaches, which may miss subtle but significant morphological changes. Here, we describe the use of embryonic Drosophila heart formation as a simplified system to quantitatively study cell matching. We provide a step-by-step protocol for large-scale embryo preparation and immunostaining and imaging details. We also describe steps for quantifying cellular mismatch from the batch images. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2018 and 2020).

The early Drosophila embryo as a model system for quantitative biology.

With the rise of new tools, from controlled genetic manipulations and optogenetics to improved microscopy, it is now possible to make clear, quantitative and reproducible measurements of biological processes. The humble fruit fly Drosophila melanogaster, with its ease of genetic manipulation combined with excellent imaging accessibility, has become a major model system for performing quantitative in vivo measurements. Such measurements are driving a new wave of interest from physicists and engineers, who are developing a range of testable dynamic models of active systems to understand fundamental biological processes. The reproducibility of the early Drosophila embryo has been crucial for understanding how biological systems are robust to unavoidable noise during development. Insights from quantitative in vivo experiments in the Drosophila embryo are having an impact on our understanding of critical biological processes, such as how cells make decisions and how complex tissue shape emerges. Here, to highlight the power of using Drosophila embryogenesis for quantitative biology, I focus on three main areas: (1) formation and robustness of morphogen gradients; (2) how gene regulatory networks ensure precise boundary formation; and (3) how mechanical interactions drive packing and tissue folding. I further discuss how such data has driven advances in modelling.

The role of cellular active stresses in shaping the zebrafish body axis.

Tissue remodelling and organ shaping during morphogenesis are products of mechanical forces generated at the cellular level. These cell-scale forces can be coordinated across the tissue via information provided by biochemical and mechanical cues. Such coordination leads to the generation of complex tissue shape during morphogenesis. In this short review, we elaborate the role of cellular active stresses in vertebrate axis morphogenesis, primarily using examples from postgastrulation development of the zebrafish embryo.

Mechanics of epidermal morphogenesis in the Drosophila pupa.

Adult epidermal development in Drosophila showcases a striking balance between en masse spreading of the developing adult precursor tissues and retraction of the degenerating larval epidermis. The adult precursor tissues, driven by both intrinsic plasticity and extrinsic mechanical cues, shape the segments of the adult epidermis and appendages. Here, we review the tissue architectural changes that occur during epidermal morphogenesis in the Drosophila pupa, with a particular emphasis on the underlying mechanical principles. We highlight recent developments in our understanding of adult epidermal morphogenesis. We further discuss the forces that drive these morphogenetic events and finally outline open questions and challenges.

Scaling of internal organs during Drosophila embryonic development.

Many species show a diverse range of sizes; for example, domestic dogs have large variation in body mass. Yet, the internal structure of the organism remains similar, i.e., the system scales to organism size. Drosophila melanogaster has been a powerful model system for exploring scaling mechanisms. In the early embryo, gene expression boundaries scale very precisely to embryo length. Later in development, the adult wings grow with remarkable symmetry and scale well with animal size. Yet, our knowledge of whether internal organs initially scale to embryo size remains largely unknown. Here, we utilize artificially small Drosophila embryos to explore how three critical internal organs-the heart, hindgut, and ventral nerve cord (VNC)-adapt to changes in embryo morphology. We find that the heart scales precisely with embryo length. Intriguingly, reduction in cardiac cell length, rather than number, appears to be important in controlling heart length. The hindgut, which is the first chiral organ to form, displays scaling with embryo size under large-scale changes in the artificially smaller embryos but shows few hallmarks of scaling within wild-type size variation. Finally, the VNC only displays weak scaling behavior; even large changes in embryo geometry result in only small shifts in VNC length. This suggests that the VNC may have an intrinsic minimal length that is largely independent of embryo length. Overall, our work shows that internal organs can adapt to embryo size changes in Drosophila, but the extent to which they scale varies significantly between organs.

Growing Up in a Changing World: Environmental Regulation of Development in Insects.

All organisms are exposed to changes in their environment throughout their life cycle. When confronted with these changes, they adjust their development and physiology to ensure that they can produce the functional structures necessary for survival and reproduction. While some traits are remarkably invariant, or robust, across environmental conditions, others show high degrees of variation, known as plasticity. Generally, developmental processes that establish cell identity are thought to be robust to environmental perturbation, while those relating to body and organ growth show greater degrees of plasticity. However, examples of plastic patterning and robust organ growth demonstrate that this is not a hard-and-fast rule.In this review, we explore how the developmental context and the gene regulatory mechanisms underlying trait formation determine the impacts of the environment on development in insects. Furthermore, we outline future issues that need to be resolved to understand how the structure of signaling networks defines whether a trait displays plasticity or robustness.

Roadmap for the multiscale coupling of biochemical and mechanical signals during development.

The way in which interactions between mechanics and biochemistry lead to the emergence of complex cell and tissue organization is an old question that has recently attracted renewed interest from biologists, physicists, mathematicians and computer scientists. Rapid advances in optical physics, microscopy and computational image analysis have greatly enhanced our ability to observe and quantify spatiotemporal patterns of signalling, force generation, deformation, and flow in living cells and tissues. Powerful new tools for genetic, biophysical and optogenetic manipulation are allowing us to perturb the underlying machinery that generates these patterns in increasingly sophisticated ways. Rapid advances in theory and computing have made it possible to construct predictive models that describe how cell and tissue organization and dynamics emerge from the local coupling of biochemistry and mechanics. Together, these advances have opened up a wealth of new opportunities to explore how mechanochemical patterning shapes organismal development. In this roadmap, we present a series of forward-looking case studies on mechanochemical patterning in development, written by scientists working at the interface between the physical and biological sciences, and covering a wide range of spatial and temporal scales, organisms, and modes of development. Together, these contributions highlight the many ways in which the dynamic coupling of mechanics and biochemistry shapes biological dynamics: from mechanoenzymes that sense force to tune their activity and motor output, to collectives of cells in tissues that flow and redistribute biochemical signals during development.

Cortical tension overrides geometrical cues to orient microtubules in confined protoplasts.

In plant cells, cortical microtubules (CMTs) generally control morphogenesis by guiding cellulose synthesis. CMT alignment has been proposed to depend on geometrical cues, with microtubules aligning with the cell long axis in silico and in vitro. Yet, CMTs are usually transverse in vivo, i.e., along predicted maximal tension, which is transverse for cylindrical pressurized vessels. Here, we adapted a microwell setup to test these predictions in a single-cell system. We confined protoplasts laterally to impose a curvature ratio and modulated pressurization through osmotic changes. We find that CMTs can be longitudinal or transverse in wallless protoplasts and that the switch in CMT orientation depends on pressurization. In particular, longitudinal CMTs become transverse when cortical tension increases. This explains the dual behavior of CMTs in planta: CMTs become longitudinal when stress levels become low, while stable transverse CMT alignments in tissues result from their autonomous response to tensile stress fluctuations.