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.

The multimodal action of G alpha q in coordinating growth and homeostasis in the Drosophila wing imaginal disc.

Background: G proteins mediate cell responses to various ligands and play key roles in organ development. Dysregulation of G-proteins or Ca 2+ signaling impacts many human diseases and results in birth defects. However, the downstream effectors of specific G proteins in developmental regulatory networks are still poorly understood. Methods: We employed the Gal4/UAS binary system to inhibit or overexpress Gαq in the wing disc, followed by phenotypic analysis. Immunohistochemistry and next-gen RNA sequencing identified the downstream effectors and the signaling cascades affected by the disruption of Gαq homeostasis. Results: Here, we characterized how the G protein subunit Gαq tunes the size and shape of the wing in the larval and adult stages of development. Downregulation of Gαq in the wing disc reduced wing growth and delayed larval development. Gαq overexpression is sufficient to promote global Ca 2+ waves in the wing disc with a concomitant reduction in the Drosophila final wing size and a delay in pupariation. The reduced wing size phenotype is further enhanced when downregulating downstream components of the core Ca 2+ signaling toolkit, suggesting that downstream Ca 2+ signaling partially ameliorates the reduction in wing size. In contrast, Gαq -mediated pupariation delay is rescued by inhibition of IP 3 R, a key regulator of Ca 2+ signaling. This suggests that Gαq regulates developmental phenotypes through both Ca 2+ -dependent and Ca 2+ -independent mechanisms. RNA seq analysis shows that disruption of Gαq homeostasis affects nuclear hormone receptors, JAK/STAT pathway, and immune response genes. Notably, disruption of Gαq homeostasis increases expression levels of Dilp8, a key regulator of growth and pupariation timing. Conclusion: Gαq activity contributes to cell size regulation and wing metamorphosis. Disruption to Gαq homeostasis in the peripheral wing disc organ delays larval development through ecdysone signaling inhibition. Overall, Gαq signaling mediates key modules of organ size regulation and epithelial homeostasis through the dual action of Ca 2+ -dependent and independent mechanisms.

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.

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.