Tools to Examine Mechanotransduction

Organisms experience acute and chronic mechanical forces. Acute mechanical stimulation in Drosophila induces the ectopic expression of developmental regulatory genes. Chronic exposure to microgravity and hypergravity causes many changes in mRNA levels and cell behavior.
These transcriptional changes occur downstream of mechanical transduction pathways yet to be identified. To investigate pathways of mechanotransduction, this thesis developed tools to apply acute and chronic mechanical stimulation in vivo with high-throughput:
• A mesofluidic device to automatic align, immobilize, compress,
image, and recover hundreds of live Drosophila embryos
• A custom centrifuge to apply hypergravity to hundreds of Drosophila
and zebrafish embryos
• A 3D-printed stamp to align zebrafish embryos for imaging
• A visual processing algorithm to segment Rohon-Beard neurons
• A series of macros to automate hyperstack and DIGE analysis
Using these methods, we:
• Show mechanical induction of twist in Drosophila
• Show gravitational induction of neurogenin in zebrafish
• Map and quantify the ectopic distribution of twist
• Measure Drosophila embryo stiffness
• Blueprint the development of future mesofluidic devices
• Run comparative proteomics across three mechanical modes, identifying
14 reciprocal changes between simulated microgravity and
hypergravity, 7 of which are shared with compression
Taken together, this thesis is a study in interdisciplinary collaboration to create a pipeline of tools. The unifying theme is high-throughput in vivo mechanotransduction. A ”mesomechanics” approach combines
the high-throughput automation and precision of microfluidics, automated image processing, and proteomics, with the biological relevance of live embryos to examine mechanotransduction. The long-term goal is to uncover and dissect pathways of mechanotransduction required
for normal cell function, development, and disease.