Farkas, J. E. | Search bioRxiv

bioRxiv2021

Local mechanical stimuli shape tissue growth in vertebrate joint morphogenesis

The correct formation of synovial joints is essential for proper motion throughout life. Movement-induced forces are critical to creating correctly shaped joints, but it is unclear how cells sense and respond to these mechanical cues. To determine how mechanical stimuli drive joint morphogenesis, we combined experiments on regenerating axolotl (Ambystoma mexicanum) forelimbs with a poroelastic model of bone rudiment growth. Animals either regrew forelimbs normally (control) or were injected with a TRPV4 agonist to impair chondrocyte mechanosensitivity during joint morphogenesis. We quantified growth and shape in regrown humeri from whole mount light sheet fluorescence images of the regenerated limbs. Results revealed statistically significant differences in morphology and cell proliferation between groups, indicating that mechanical stimuli play a role in the shaping of joints. We simulated local tissue growth in a finite element model with a biological contribution to growth proportional to chondrocyte density, and a mechanical contribution to growth proportional to fluid pressure. Computational predictions agreed with experimental outcomes, suggesting that interstitial pressure driven from cyclic mechanical stimuli promotes local tissue growth. Predictive computational models informed by experimental findings allow us to explore potential physical mechanisms involved in tissue growth to advance our understanding of the mechanobiology of joint morphogenesis.

developmental biology↗

bioRxiv2020

3D Visualization of Macromolecule Synthesis

Measuring nascent macromolecular synthesis in vivo is key to understanding how cells and tissues progress through development and respond to external cues. Here, we perform in vivo injection of alkyne- or azide-modified analogs of thymidine, uridine, methionine, and glucosamine to label nascent synthesis of DNA, RNA, protein, and glycosylation. Three-dimensional volumetric imaging of nascent macromolecule synthesis was performed in axolotl salamander tissue using whole mount click chemistry-based fluorescent staining followed by light sheet fluorescent microscopy. We also developed an image processing pipeline for segmentation and classification of morphological regions of interest and individual cells, and we apply this pipeline to the regenerating humerus. We demonstrate our approach is sensitive to biological perturbations by measuring changes in DNA synthesis after limb denervation. This method provides a powerful means to quantitatively interrogate macromolecule synthesis in heterogenous tissues at the organ, cellular, and molecular levels of organization.

developmental biology↗