Calcium Imaging in Drosophila.

Ex vivo calcium imaging in Drosophila opens an expansive amount of research avenues for the study of live signal propagation through complex tissue. Here, we describe how to isolate Drosophila organs of interest, like the developing wing imaginal disc and larval brain, culture them for extended periods, up to 10 h, and how to image the calcium dynamics occurring within them using genetically encoded biosensors like GCaMP. This protocol enables the study of complex calcium signaling dynamics, which is conserved throughout biology in such processes as cell differentiation and proliferation, immune reactions, wound healing, and cell-to-cell and organ-to-organ communication, among others. These methods also allow pharmacological compounds to be tested to observe effects on calcium dynamics with the applications of target identification and therapeutic development.

Low-cost Polyethylene Terephthalate Lamination Microfluidics Designs for Multiplexed Zebrafish Imaging.

Zebrafish embryos are transparent and thus uniquely suited for noninvasive intravital imaging of fundamental processes, such as wound healing and immune cell migration. Microfluidic devices are used for entrapment to support long-term imaging of multicellular organisms, including zebrafish. However, the fabrication of these devices using soft lithography requires specialized facilities and competency in 3D printing, which may not be accessible to every lab. Our adaptation of a previously developed low-cost polyethylene terephthalate lamination method for constructing microfluidic devices increases accessibility by enabling design fabrication and iteration for a fraction of the technical investment of conventional techniques. We use a device made with this method, the Rotational Assistant for Danio Imaging of Subsequent Healing (RADISH), to accommodate drug treatment, manual wounding, and long-term imaging of up to four embryos in the same field of view. With this new design, we successfully capture gross morphological characteristics of the calcium signal around laser ablation and manual transection wounds for multiple embryos in the 2 h immediately following injury, as well as neutrophil recruitment to the wound edge for 24 h.

Mechanical Compression of Drosophila Embryos Using Rapid Fabrication Microfluidic Devices.

Microfluidic devices support developmental and mechanobiology studies by enabling the precise control of electrical, chemical, and mechanical stimuli at the microscale. Here, we describe the fabrication of customizable microfluidic devices and demonstrate their efficacy in applying mechanical loads to micro-organs and whole organisms, such as Drosophila embryos. The fabrication technique consists in the use of xurography to define channels and chambers using thin layers of thermoplastics and glass. The superposition of layers followed by thermal lamination produces robust and reproducible devices that are easily adapted for a variety of experiments. The integration of deformable layers and glass in these devices facilitates the imaging of cellular and molecular dynamics in biological specimens under mechanical loads. The method is highly adaptable for studies in mechanobiology.

Proteins clump: Mechanics and transport during neurodegeneration.

This summary of recent contributions in the Biophysical Journal from 2020 to 2023 highlights new mechanistic insights into key biomechanical and biophysical aspects of neurodegeneration. Neurodegeneration encompasses complex diseases characterized by the progressive loss of neuronal function, often linked to protein accumulation and aggregation. Several factors, including mechanical properties and structural composition of brain tissue, formation of proteinaceous condensates within cells, and protein transport between cells, impact the loss of neural function.

Piezo regulates epithelial topology and promotes precision in organ size control.

Mechanosensitive Piezo channels regulate cell division, cell extrusion, and cell death. However, systems-level functions of Piezo in regulating organogenesis remain poorly understood. Here, we demonstrate that Piezo controls epithelial cell topology to ensure precise organ growth by integrating live-imaging experiments with pharmacological and genetic perturbations and computational modeling. Notably, the knockout or knockdown of Piezo increases bilateral asymmetry in wing size. Piezo's multifaceted functions can be deconstructed as either autonomous or non-autonomous based on a comparison between tissue-compartment-level perturbations or between genetic perturbation populations at the whole-tissue level. A computational model that posits cell proliferation and apoptosis regulation through modulation of the cutoff tension required for Piezo channel activation explains key cell and tissue phenotypes arising from perturbations of Piezo expression levels. Our findings demonstrate that Piezo promotes robustness in regulating epithelial topology and is necessary for precise organ size control.

Reverse engineering morphogenesis through Bayesian optimization of physics-based models.

Morphogenetic programs coordinate cell signaling and mechanical interactions to shape organs. In systems and synthetic biology, a key challenge is determining optimal cellular interactions for predicting organ shape, size, and function. Physics-based models defining the subcellular force distribution facilitate this, but it is challenging to calibrate parameters in these models from data. To solve this inverse problem, we created a Bayesian optimization framework to determine the optimal cellular force distribution such that the predicted organ shapes match the experimentally observed organ shapes. This integrative framework employs Gaussian Process Regression, a non-parametric kernel-based probabilistic machine learning modeling paradigm, to learn the mapping functions relating to the morphogenetic programs that maintain the final organ shape. We calibrated and tested the method on Drosophila wing imaginal discs to study mechanisms that regulate epithelial processes ranging from development to cancer. The parameter estimation framework successfully infers the underlying changes in core parameters needed to match simulation data with imaging data of wing discs perturbed with collagenase. The computational pipeline identifies distinct parameter sets mimicking wild-type shapes. It enables a global sensitivity analysis to support the regulation of actomyosin contractility and basal ECM stiffness to generate and maintain the curved shape of the wing imaginal disc. The optimization framework, combined with experimental imaging, identified that Piezo, a mechanosensitive ion channel, impacts fold formation by regulating the apical-basal balance of actomyosin contractility and elasticity of ECM. This workflow is extensible toward reverse-engineering morphogenesis across organ systems and for real-time control of complex multicellular systems.

Balancing competing effects of tissue growth and cytoskeletal regulation during Drosophila wing disc development.

How a developing organ robustly coordinates the cellular mechanics and growth to reach a final size and shape remains poorly understood. Through iterations between experiments and model simulations that include a mechanistic description of interkinetic nuclear migration, we show that the local curvature, height, and nuclear positioning of cells in the Drosophila wing imaginal disc are defined by the concurrent patterning of actomyosin contractility, cell-ECM adhesion, ECM stiffness, and interfacial membrane tension. We show that increasing cell proliferation via different growth-promoting pathways results in two distinct phenotypes. Triggering proliferation through insulin signaling increases basal curvature, but an increase in growth through Dpp signaling and Myc causes tissue flattening. These distinct phenotypic outcomes arise from differences in how each growth pathway regulates the cellular cytoskeleton, including contractility and cell-ECM adhesion. The coupled regulation of proliferation and cytoskeletal regulators is a general strategy to meet the multiple context-dependent criteria defining tissue morphogenesis.

Quantitative insights in tissue growth and morphogenesis with optogenetics.

Cells communicate with each other to jointly regulate cellular processes during cellular differentiation and tissue morphogenesis. This multiscale coordination arises through the spatiotemporal activity of morphogens to pattern cell signaling and transcriptional factor activity. This coded information controls cell mechanics, proliferation, and differentiation to shape the growth and morphogenesis of organs. While many of the molecular components and physical interactions have been identified in key model developmental systems, there are still many unresolved questions related to the dynamics involved due to challenges in precisely perturbing and quantitatively measuring signaling dynamics. Recently, a broad range of synthetic optogenetic tools have been developed and employed to quantitatively define relationships between signal transduction and downstream cellular responses. These optogenetic tools can control intracellular activities at the single cell or whole tissue scale to direct subsequent biological processes. In this brief review, we highlight a selected set of studies that develop and implement optogenetic tools to unravel quantitative biophysical mechanisms for tissue growth and morphogenesis across a broad range of biological systems through the manipulation of morphogens, signal transduction cascades, and cell mechanics. More generally, we discuss how optogenetic tools have emerged as a powerful platform for probing and controlling multicellular development.

MAPPER: An Open-Source, High-Dimensional Image Analysis Pipeline Unmasks Differential Regulation of Drosophila Wing Features.

Phenomics requires quantification of large volumes of image data, necessitating high throughput image processing approaches. Existing image processing pipelines for Drosophila wings, a powerful genetic model for studying the underlying genetics for a broad range of cellular and developmental processes, are limited in speed, precision, and functional versatility. To expand on the utility of the wing as a phenotypic screening system, we developed MAPPER, an automated machine learning-based pipeline that quantifies high-dimensional phenotypic signatures, with each dimension quantifying a unique morphological feature of the Drosophila wing. MAPPER magnifies the power of Drosophila phenomics by rapidly quantifying subtle phenotypic differences in sample populations. We benchmarked MAPPER's accuracy and precision in replicating manual measurements to demonstrate its widespread utility. The morphological features extracted using MAPPER reveal variable sexual dimorphism across Drosophila species and unique underlying sex-specific differences in morphogen signaling in male and female wings. Moreover, the length of the proximal-distal axis across the species and sexes shows a conserved scaling relationship with respect to the wing size. In sum, MAPPER is an open-source tool for rapid, high-dimensional analysis of large imaging datasets. These high-content phenomic capabilities enable rigorous and systematic identification of genotype-to-phenotype relationships in a broad range of screening and drug testing applications and amplify the potential power of multimodal genomic approaches.

From spikes to intercellular waves: Tuning intercellular calcium signaling dynamics modulates organ size control.

Information flow within and between cells depends significantly on calcium (Ca2+) signaling dynamics. However, the biophysical mechanisms that govern emergent patterns of Ca2+ signaling dynamics at the organ level remain elusive. Recent experimental studies in developing Drosophila wing imaginal discs demonstrate the emergence of four distinct patterns of Ca2+ activity: Ca2+ spikes, intercellular Ca2+ transients, tissue-level Ca2+ waves, and a global "fluttering" state. Here, we used a combination of computational modeling and experimental approaches to identify two different populations of cells within tissues that are connected by gap junction proteins. We term these two subpopulations "initiator cells," defined by elevated levels of Phospholipase C (PLC) activity, and "standby cells," which exhibit baseline activity. We found that the type and strength of hormonal stimulation and extent of gap junctional communication jointly determine the predominate class of Ca2+ signaling activity. Further, single-cell Ca2+ spikes are stimulated by insulin, while intercellular Ca2+ waves depend on Gαq activity. Our computational model successfully reproduces how the dynamics of Ca2+ transients varies during organ growth. Phenotypic analysis of perturbations to Gαq and insulin signaling support an integrated model of cytoplasmic Ca2+ as a dynamic reporter of overall tissue growth. Further, we show that perturbations to Ca2+ signaling tune the final size of organs. This work provides a platform to further study how organ size regulation emerges from the crosstalk between biochemical growth signals and heterogeneous cell signaling states.

Pinching and pushing: fold formation in the Drosophila dorsal epidermis.

Epithelial folding is a fundamental morphogenetic process that shapes planar epithelial sheets into complex three-dimensional structures. Multiple mechanisms can generate epithelial folds, including apical constriction, which acts locally at the cellular level, differential growth on the tissue scale, or buckling because of compression from neighboring tissues. Here, we investigate the formation of dorsally located epithelial folds at segment boundaries during the late stages of Drosophila embryogenesis. We found that the fold formation at the segment boundaries occurs through the juxtaposition of two key morphogenetic processes: local apical constriction and tissue-level compressive forces from posterior segments. Further, we found that epidermal spreading and fold formation are accompanied by spatiotemporal pulses of Hedgehog (Hh) signaling. A computational model that incorporates the local forces generated from the differential tensions of the apical, basal, and lateral sides of the cell and active forces generated within the whole tissue recapitulates the overall fold formation process in wild-type and Hh overexpression conditions. In sum, this work demonstrates how epithelial folding depends on multiple, separable physical mechanisms to generate the final morphology of the dorsal epidermis. This work illustrates the modularity of morphogenetic unit operations that occur during epithelial morphogenesis.

Epithelial organ shape is generated by patterned actomyosin contractility and maintained by the extracellular matrix.

Epithelial sheets define organ architecture during development. Here, we employed an iterative multiscale computational modeling and quantitative experimental approach to decouple direct and indirect effects of actomyosin-generated forces, nuclear positioning, extracellular matrix, and cell-cell adhesion in shaping Drosophila wing imaginal discs. Basally generated actomyosin forces generate epithelial bending of the wing disc pouch. Surprisingly, acute pharmacological inhibition of ROCK-driven actomyosin contractility does not impact the maintenance of tissue height or curved shape. Computational simulations show that ECM tautness provides only a minor contribution to modulating tissue shape. Instead, passive ECM pre-strain serves to maintain the shape independent from actomyosin contractility. These results provide general insight into how the subcellular forces are generated and maintained within individual cells to induce tissue curvature. Thus, the results suggest an important design principle of separable contributions from ECM prestrain and actomyosin tension during epithelial organogenesis and homeostasis.

Rab11b-mediated integrin recycling promotes brain metastatic adaptation and outgrowth.

Breast cancer brain metastases (BCBM) have a 5-20 year latency and account for 30% of mortality; however, mechanisms governing adaptation to the brain microenvironment remain poorly defined. We combine time-course RNA-sequencing of BCBM development with a Drosophila melanogaster genetic screen, and identify Rab11b as a functional mediator of metastatic adaptation. Proteomic analysis reveals that Rab11b controls the cell surface proteome, recycling proteins required for successful interaction with the microenvironment, including integrin ß1. Rab11b-mediated control of integrin ß1 surface expression allows efficient engagement with the brain ECM, activating mechanotransduction signaling to promote survival. Lipophilic statins prevent membrane association and activity of Rab11b, and we provide proof-of principle that these drugs prevent breast cancer adaptation to the brain microenvironment. Our results identify Rab11b-mediated recycling of integrin ß1 as regulating BCBM, and suggest that the recycleome, recycling-based control of the cell surface proteome, is a previously unknown driver of metastatic adaptation and outgrowth.

Rapid Fabrication of Custom Microfluidic Devices for Research and Educational Applications.

Microfluidic devices allow for the manipulation of fluids, particles, cells, micro-sized organs or organisms in channels ranging from the nano to submillimeter scales. A rapid increase in the use of this technology in the biological sciences has prompted a need for methods that are accessible to a wide range of research groups. Current fabrication standards, such as PDMS bonding, require expensive and time consuming lithographic and bonding techniques. A viable alternative is the use of equipment and materials that are easily affordable, require minimal expertise and allow for the rapid iteration of designs. In this work we describe a protocol for designing and producing PET-laminates (PETLs), microfluidic devices that are inexpensive, easy to fabricate, and consume significantly less time to generate than other approaches to microfluidics technology. They consist of thermally bonded film sheets, in which channels and other features are defined using a craft cutter. PETLs solve field-specific technical challenges while dramatically reducing obstacles to adoption. This approach facilitates the accessibility of microfluidics devices in both research and educational settings, providing a reliable platform for new methods of inquiry.

Microfluidics on the fly: Inexpensive rapid fabrication of thermally laminated microfluidic devices for live imaging and multimodal perturbations of multicellular systems.

Microfluidic devices provide a platform for analyzing both natural and synthetic multicellular systems. Currently, substantial capital investment and expertise are required for creating microfluidic devices using standard soft-lithography. These requirements present barriers to entry for many nontraditional users of microfluidics, including developmental biology laboratories. Therefore, fabrication methodologies that enable rapid device iteration and work "out-of-the-box" can accelerate the integration of microfluidics with developmental biology. Here, we have created and characterized low-cost hybrid polyethylene terephthalate laminate (PETL) microfluidic devices that are suitable for cell and micro-organ culture assays. These devices were validated with mammalian cell lines and the Drosophila wing imaginal disc as a model micro-organ. First, we developed and tested PETLs that are compatible with both long-term cultures and high-resolution imaging of cells and organs. Further, we achieved spatiotemporal control of chemical gradients across the wing discs with a multilayered microfluidic device. Finally, we created a multilayered device that enables controllable mechanical loading of micro-organs. This mechanical actuation assay was used to characterize the response of larval wing discs at different developmental stages. Interestingly, increased deformation of the older wing discs for the same mechanical loading suggests that the compliance of the organ is increased in preparation for subsequent morphogenesis. Together, these results demonstrate the applicability of hybrid PETL devices for biochemical and mechanobiology studies on micro-organs and provide new insights into the mechanics of organ development.

Tools to reverse-engineer multicellular systems: case studies using the fruit fly.

Reverse-engineering how complex multicellular systems develop and function is a grand challenge for systems bioengineers. This challenge has motivated the creation of a suite of bioengineering tools to develop increasingly quantitative descriptions of multicellular systems. Here, we survey a selection of these tools including microfluidic devices, imaging and computer vision techniques. We provide a selected overview of the emerging cross-talk between engineering methods and quantitative investigations within developmental biology. In particular, the review highlights selected recent examples from the Drosophila system, an excellent platform for understanding the interplay between genetics and biophysics. In sum, the integrative approaches that combine multiple advances in these fields are increasingly necessary to enable a deeper understanding of how to analyze both natural and synthetic multicellular systems.

Decoding Calcium Signaling Dynamics during Drosophila Wing Disc Development.

The robust specification of organ development depends on coordinated cell-cell communication. This process requires signal integration among multiple pathways, relying on second messengers such as calcium ions. Calcium signaling encodes a significant portion of the cellular state by regulating transcription factors, enzymes, and cytoskeletal proteins. However, the relationships between the inputs specifying cell and organ development, calcium signaling dynamics, and final organ morphology are poorly understood. Here, we have designed a quantitative image-analysis pipeline for decoding organ-level calcium signaling. With this pipeline, we extracted spatiotemporal features of calcium signaling dynamics during the development of the Drosophila larval wing disc, a genetic model for organogenesis. We identified specific classes of wing phenotypes that resulted from calcium signaling pathway perturbations, including defects in gross morphology, vein differentiation, and overall size. We found four qualitative classes of calcium signaling activity. These classes can be ordered based on agonist stimulation strength Gαq-mediated signaling. In vivo calcium signaling dynamics depend on both receptor tyrosine kinase/phospholipase C γ and G protein-coupled receptor/phospholipase C ß activities. We found that spatially patterned calcium dynamics correlate with known differential growth rates between anterior and posterior compartments. Integrated calcium signaling activity decreases with increasing tissue size, and it responds to morphogenetic perturbations that impact organ growth. Together, these findings define how calcium signaling dynamics integrate upstream inputs to mediate multiple response outputs in developing epithelial organs.

Calcium as a signal integrator in developing epithelial tissues.

Decoding how tissue properties emerge across multiple spatial and temporal scales from the integration of local signals is a grand challenge in quantitative biology. For example, the collective behavior of epithelial cells is critical for shaping developing embryos. Understanding how epithelial cells interpret a diverse range of local signals to coordinate tissue-level processes requires a systems-level understanding of development. Integration of multiple signaling pathways that specify cell signaling information requires second messengers such as calcium ions. Increasingly, specific roles have been uncovered for calcium signaling throughout development. Calcium signaling regulates many processes including division, migration, death, and differentiation. However, the pleiotropic and ubiquitous nature of calcium signaling implies that many additional functions remain to be discovered. Here we review a selection of recent studies to highlight important insights into how multiple signals are transduced by calcium transients in developing epithelial tissues. Quantitative imaging and computational modeling have provided important insights into how calcium signaling integration occurs. Reverse-engineering the conserved features of signal integration mediated by calcium signaling will enable novel approaches in regenerative medicine and synthetic control of morphogenesis.

Preclinical study of a Kv11.1 potassium channel activator as antineoplastic approach for breast cancer.

Potassium ion (K+) channels have been recently found to play a critical role in cancer biology. Despite that pharmacologic manipulation of ion channels is recognized as an important therapeutic approach, very little is known about the effects of targeting of K+ channels in cancer. In this study, we demonstrate that use of the Kv11.1 K+ channel activator NS1643 inhibits tumor growth in an in vivo model of breast cancer. Tumors exposed to NS1643 had reduced levels of proliferation markers, high expression levels of senescence markers, increased production of ROS and DNA damage compared to tumors of untreated mice. Importantly, mice treated with NS1643 did not exhibit significant cardiac dysfunction. In conclusion, pharmacological stimulation of Kv11.1 activity produced arrested TNBC-derived tumor growth by generating DNA damage and senescence without significant side effects. We propose that use of Kv11.1 channels activators could be considered as a possible pharmacological strategy against breast tumors.

A NEW REGISTRATION APPROACH FOR DYNAMIC ANALYSIS OF CALCIUM SIGNALS IN ORGANS.

Wing disc pouches of fruit flies are a powerful genetic model for studying physiological intercellular calcium (Ca 2+) signals for dynamic analysis of cell signaling in organ development and disease studies. A key to analyzing spatial-temporal patterns of Ca 2+ signal waves is to accurately align the pouches across image sequences. However, pouches in different image frames may exhibit extensive intensity oscillations due to Ca 2+ signaling dynamics, and commonly used multimodal non-rigid registration methods may fail to achieve satisfactory results. In this paper, we develop a new two-phase non-rigid registration approach to register pouches in image sequences. First, we conduct segmentation of the region of interest. (i.e., pouches) using a deep neural network model. Second, we use a B-spline based registration to obtain an optimal transformation and align pouches across the image sequences. Evaluated using both synthetic data and real pouch data, our method considerably outperforms the state-of-the-art non-rigid registration methods.