Robust cell tracking in epithelial tissues through identification of maximum common subgraphs

Tracking of cells in live-imaging microscopy videos of epithelial sheets is a powerful tool for investigating fundamental processes in embryonic development. Characterizing cell growth, proliferation, intercalation and apoptosis in epithelia helps us to understand how morphogenetic processes such as tissue invagination and extension are locally regulated and controlled. Accurate cell tracking requires correctly resolving cells entering or leaving the field of view between frames, cell neighbour exchanges, cell removals and cell divisions. However, current tracking methods for epithelial sheets are not robust to large morphogenetic deformations and require significant manual interventions. Here, we present a novel algorithm for epithelial cell tracking, exploiting the graph-theoretic concept of a ‘maximum common subgraph’ to track cells between frames of a video. Our algorithm does not require the adjustment of tissue-specific parameters, and scales in sub-quadratic time with tissue size. It does not rely on precise positional information, permitting large cell movements between frames and enabling tracking in datasets acquired at low temporal resolution due to experimental constraints such as phototoxicity. To demonstrate the method, we perform tracking on the Drosophila embryonic epidermis and compare cell–cell rearrangements to previous studies in other tissues. Our implementation is open source and generally applicable to epithelial tissues

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

Patterning of wound-induced intercellular Ca2+ flashes in a developing epithelium

Differential mechanical force distributions are increasingly recognized to provide important feedback into the control of an organ's final size and shape. As a second messenger that integrates and relays mechanical information to the cell, calcium ions (Ca2+) are a prime candidate for providing important information on both the overall mechanical state of the tissue and resulting behavior at the individual-cell level during development. Still, how the spatiotemporal properties of Ca2+ transients reflect the underlying mechanical characteristics of tissues is still poorly understood. Here we use an established model system of an epithelial tissue, the Drosophila wing imaginal disc, to investigate how tissue properties impact the propagation of Ca2+ transients induced by laser ablation. The resulting intercellular Ca2+ flash is found to be mediated by inositol 1,4,5-trisphosphate and depends on gap junction communication. Further, we find that intercellular Ca2+ transients show spatially non-uniform characteristics across the proximal–distal axis of the larval wing imaginal disc, which exhibit a gradient in cell size and anisotropy. A computational model of Ca2+ transients is employed to identify the principle factors explaining the spatiotemporal patterning dynamics of intercellular Ca2+ flashes. The relative Ca2+ flash anisotropy is principally explained by local cell shape anisotropy. Further, Ca2+ velocities are relatively uniform throughout the wing disc, irrespective of cell size or anisotropy. This can be explained by the opposing effects of cell diameter and cell elongation on intercellular Ca2+ propagation. Thus, intercellular Ca2+ transients follow lines of mechanical tension at velocities that are largely independent of tissue heterogeneity and reflect the mechanical state of the underlying tissue

Principles for the design of multicellular engineered living systems

Remarkable progress in bioengineering over the past two decades has enabled the formulation of fundamental design principles for a variety of medical and non-medical applications. These advancements have laid the foundation for building multicellular engineered living systems (M-CELS) from biological parts, forming functional modules integrated into living machines. These cognizant design principles for living systems encompass novel genetic circuit manipulation, self-assembly, cell–cell/matrix communication, and artificial tissues/organs enabled through systems biology, bioinformatics, computational biology, genetic engineering, and microfluidics. Here, we introduce design principles and a blueprint for forward production of robust and standardized M-CELS, which may undergo variable reiterations through the classic design-build-test-debug cycle. This Review provides practical and theoretical frameworks to forward-design, control, and optimize novel M-CELS. Potential applications include biopharmaceuticals, bioreactor factories, biofuels, environmental bioremediation, cellular computing, biohybrid digital technology, and experimental investigations into mechanisms of multicellular organisms normally hidden inside the “black box” of living cells

Bistability coordinates activation of the EGFR and DPP pathways in Drosophila vein differentiation

Cell differentiation in developing tissues is controlled by a small set of signaling pathways, which must coordinate the timing and levels of activation to ensure robust and precise outcomes. Highly coordinated activation of signaling pathways can result from cross-regulatory interactions in multi-pathway networks. Here we explore the dynamics and function of pathway coordination between the EGFR and DPP pathways during Drosophila wing-vein differentiation. We show that simultaneous activation of both the EGFR and DPP pathways must be maintained for vein cell differentiation and that above-threshold ectopic activation of either pathway is sufficient to drive vein cell differentiation outside the proveins. The joint activation of the EGFR and DPP signaling systems is ensured by a positive feedback loop, in which the two pathways stimulate each other at the level of ligand production

Capabilities and Limitations of Tissue Size Control through Passive Mechanical Forces

Embryogenesis is an extraordinarily robust process, exhibiting the ability to control tissue size and repair patterning defects in the face of environmental and genetic perturbations. The size and shape of a developing tissue is a function of the number and size of its constituent cells as well as their geometric packing. How these cellular properties are coordinated at the tissue level to ensure developmental robustness remains a mystery; understanding this process requires studying multiple concurrent processes that make up morphogenesis, including the spatial patterning of cell fates and apoptosis, as well as cell intercalations. In this work, we develop a computational model that aims to understand aspects of the robust pattern repair mechanisms of the Drosophila embryonic epidermal tissues. Size control in this system has previously been shown to rely on the regulation of apoptosis rather than proliferation; however, to date little work has been done to understand the role of cellular mechanics in this process. We employ a vertex model of an embryonic segment to test hypotheses about the emergence of this size control. Comparing the model to previously published data across wild type and genetic perturbations, we show that passive mechanical forces suffice to explain the observed size control in the posterior (P) compartment of a segment. However, observed asymmetries in cell death frequencies across the segment are demonstrated to require patterning of cellular properties in the model. Finally, we show that distinct forms of mechanical regulation in the model may be distinguished by differences in cell shapes in the P compartment, as quantified through experimentally accessible summary statistics, as well as by the tissue recoil after laser ablation experiments

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

Abstract 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