Shining Light on Calcium-Mediated Morphogenesis: Forward Engineering Organ Development with Optogenetics and Mechanosensation

Introduction:: Cells communicate to coordinate cellular processes across tissues, and calcium (Ca²⁺) ions are second messengers facilitating such multiscale coordination. Ca²⁺-selective cation channels mediate Ca²⁺-influx from the extracellular environment into the cytosol upon activation. However, current understanding of the biological mechanisms precludes direct control of Ca²⁺-mediated processes. Here, optogenetic and mechanosensitive channels, CsChrimson¹ and Piezo², respectively, are utilized as tools to investigate how Ca²⁺-dynamics impact epithelial growth and morphogenesis. Drosophila melanogaster was used as an in vivo system, whose wing is acutely sensitive to changes in developmental pathways. We outline the phenotypic results of controlled stimulation of CsChrimson and Piezo in Drosophila. Optogenetics has emerged as a rapidly expanding solution to better decipher biological processes as it leverages engineered light-sensitive protein constructs to control cells and their governing biomolecular processes and signaling pathways. Hence, an artificial plasma-membrane embedded channelrhodopsin, CsChrimson was used in transgenic fruit flies. Next, we used the mechanosensitive Piezo channel which is endogenously present in both humans and Drosophila. It can be activated through genetic overexpression, controlled mechanical force, or its specific agonist, Yoda1.

We demonstrate that these channels regulate cytosolic Ca²⁺ dynamics which impacts the activities of downstream proteins and growth-related biological pathways. We used optogenetic and mechanosensitive channels to forward engineer growth control in epithelia as a bottom-up approach toward tissue engineering. These findings regarding how Ca²⁺-signaling dynamics tune the robustness of organ growth and cellular processes provide scope for future applications related to phenotypic drug screening assays and innovation in treatments of human diseases.


Materials and Methods:: GAL4-UAS³, a bipartite method of activating gene expression Drosophila was used to express the gene of interest. Flies were grown at 25° C.

To ensure longer periods of viability during ex vivo imaging and allow the addition of a constant flow of media, a hybrid polyethylene terephthalate laminate (PETL) microfluidic device⁴ was used for live imaging. Standard protocol⁵ of immunohistochemistry was followed to perform antibody staining of fixed wing discs.

The wing imaginal discs were imaged using a Nikon A1R-MP laser scanning confocal microscope. Wing discs were imaged throughout the entire depth of z-planes with a step size of 0.8 μm, depending on sample thickness, with a 40x and 60x oil objective with 200 ms exposure time, and 50 nW, 405 nm, 488 nm, 561 nm, and 640 nm laser exposure at 44% laser intensity.

For optogenetic experimentation, 1 mM All-Trans-Retinal was supplemented for the functional generation of CsChrimson in Drosophila (Fig.1). The crosses were set up in darkness since white light can desensitize and deactivate the optogenetic channels. The dark condition was maintained by wrapping the vials in aluminum foil. On collecting the embryos in darkness, red light (~600 nm) was used for 12 hr/day activating CsChrimson using LED strips at the 1st instar larval stage. An Arduino microcontroller appended with transistors for amplification and resistors for dimming light was powered by a 9 V DC battery and programmed to maintain the period of illuminance. A digital illuminance meter was used to measure the level of light in lux.


Results, Conclusions, and Discussions:: We discovered that CsChrimson and Piezo can modulate cellular processes involved in morphogenesis. We first confirmed that these channels transport Ca²⁺ by using a GCaMP6f Ca²⁺ indicator in the wing discs and observed Ca²⁺ spiking dynamics for activation of both channels through light or Yoda1-mediated activation (Fig.2). This conclusively proved that CsChrimson or Piezo can regulate Ca²⁺ dynamics in the tissue. The morphology of the terminal organ for ovexpression of CsChrimson or downregulation of Piezo had severe defects in the final shape and size of the wing while overexpression of Piezo also resulted in smaller-sized wings compared to control (Fig.3,4). For CsChrimson, the adult wing phenotypes were qualitatively and quantitatively correlated with the gradual increase in the intensity of light. Surprisingly, on quantifying the area of wings for different intensities of light activation, we found that at 5 lux of light, the size of the wings was larger compared to other intensities indicating an optimum level of light and Ca²⁺ level that induces growth (Fig.4). Different phenotypes for Piezo expression levels also point to similar regulation of morphogenetic processes through regulation of Ca²⁺ level. We followed this up by investigating cellular processes and found increased cell death in wing discs for optogenetic activation for long periods and genetic overexpression or downregulation of Piezo. Interestingly, we also observed higher proliferation for overexpression of Piezo or a lower frequency of light activation (1 min on/off period) indicating a probable apoptosis-induced compensatory proliferation phenomenon (Fig.5). We hypothesize that by controlling Ca²⁺, the tissue growth can be directly controlled and the optimum level of cytosolic Ca²⁺ to promote growth can be established. Systematically varying the width-half-max of optogenetic activation, e.g., by using a lower intensity of light or a faster optogenetic activation cycle, or by changing the mechanical activation level of Piezo will determine such “Goldilocks zone” of Ca²⁺-level.

These results provide novel insights into Ca²⁺-regulated morphogenesis while raising new questions regarding downstream pathways. A new avenue of therapeutic discovery can be explored via activation period or activation level-based screening using light or mechanical activation of channels like CsChrimson or Piezo for Ca²⁺-channelopathies.


Acknowledgements (Optional): : Multiple figures were created with BioRender.com. Thanks to Dr. Sara Cole from Notre Dame Integrated Imaging Facility for helping with imaging. Thanks to Dr. Maria Unger, Dr. Francisco Huizar, Dr. Marycruz Flores Flores, Dr. Vijay Kumar Naidu Velgala, Dr, Megan Levis, David Gazzo, and Gabriel Miranda for their insights and feedback. The work was supported by NIH 1R35GM124935-01, NSF CBET-2225601 (RECODE), PTX-Purdue University 10001908-020 (EMBRIO).



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