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 Gaq-mediated signaling. In vivo calcium signaling dynamics depend on both receptor tyrosine kinase/phospholipase C g and G protein-coupled receptor/phospholipase C b 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.
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.
Ranging from miniaturized biological robots to organoids, multi-cellular engineered living systems (M-CELS) pose complex ethical and societal challenges. Some of these challenges, such as how to best distribute risks and benefits, are likely to arise in the development of any new technology. Other challenges arise specifically because of the particular characteristics of M-CELS. For example, as an engineered living system becomes increasingly complex, it may provoke societal debate about its moral considerability, perhaps necessitating protection from harm or recognition of positive moral and legal rights, particularly if derived from cells of human origin. The use of emergence-based principles in M-CELS development may also create unique challenges, making the technology difficult to fully control or predict in the laboratory as well as in applied medical or environmental settings. In response to these challenges, we argue that the M-CELS community has an obligation to systematically address the ethical and societal aspects of research and to seek input from and accountability to a broad range of stakeholders and publics. As a newly developing field, M-CELS has a significant opportunity to integrate ethically responsible norms and standards into its research and development practices from the start. With the aim of seizing this opportunity, we identify two general kinds of salient ethical issues arising from M-CELS research, and then present a set of commitments to and strategies for addressing these issues. If adopted, these commitments and strategies would help define M-CELS as not only an innovative field, but also as a model for responsible research and engineering.
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 microorgans and provide new insights into the mechanics of organ development.
Recently, organ-scale intercellular Ca 2+ transients (ICTs) were reported in the Drosophila wing disc. However, the functional in vivo significance of ICTs remains largely unknown. Here we demonstrate the in vivo relevance of intercellular Ca 2+ signaling and its impact on wing development. We report that Ca 2+ signaling in vivo decreases as wing discs mature. Ca 2+ signaling ex vivo responds to fly extract in a dose-dependent manner. This suggests ICTs occur in vivo due to chemical stimulus that varies in concentration during development. RNAi mediated inhibition of genes required for ICTs results in defects in the size, shape, and vein patterning of adult wings. It also leads to reduction or elimination of in vivo Ca 2+ transients. Further, perturbations to the extracellular matrix along the basal side of the wing disc stimulates intercellular Ca 2+ waves. This is the first identified chemically defined, non-wounding stimulus of ICTs. Together, these results point toward specific in vivo functions of intercellular Ca 2+ signaling to mediate mechanical stress dissipation and ensure robust patterning during development.
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