Simultaneous in vivo time-lapse stiffness mapping and fluorescence imaging of developing tissue

Abstract

Tissue mechanics is important for development; however, the spatio-temporal dynamics of in vivo tissue stiffness is still poorly understood. We here developed tiv-AFM, combining time-lapse in vivo atomic force microscopy with upright fluorescence imaging of embryonic tissue, to show that in the developing Xenopus brain, a stiffness gradient evolves over time because of differential cell proliferation. Subsequently, axons turn to follow this gradient, underpinning the importance of timeresolved mechanics measurements.. CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 1).A fluorescence zoom stereomicroscope equipped with an sCMOS camera (quantum yield 82%)were custom-fitted above a bio-AFM set-up ( Supplementary Fig. 1), which had a transparent pathway along the area of the cantilever. To cope with the long working distances required for imaging through the AFM head, the microscope was fitted with a 0.125 NA / 114 mm WD objective.The AFM was set up on an automated motorised stage containing a temperature-controlled sample holder to maintain live specimens at optimal conditions during the experimental time course. (Fig. 1a, b) (see online methods for details).We tested tiv-AFM using the developing Xenopus embryo brain during outgrowth of the optic tract (OT) as a model (Fig. 1c). In the OT, retinal ganglion cell axons grow in a bundle across the brain surface, making a stereotypical turn in the caudal direction en route that directs them to their target, the visual centre of the brain 13 . We previously demonstrated that by later stages of OT outgrowth (i.e. when axons had reached their target), a local stiffness gradient lies orthogonal to the path of OT axons, with the stiffer region rostral to the OT and softer region caudal to it 2 . This gradient strongly correlated with axon turning, with the OT routinely turning caudally towards softer tissue 2 .We therefore wanted to determine when this stiffness gradient first developed, whether its emergence preceded OT axon turning, and what the origin of that stiffness gradient was.To answer these questions, we performed iterated tiv-AFM measurements of the embryonic brain in vivo at early-intermediate stages, i.e. just before and during turn initiation by the first 'pioneer' OT axons. The apparent elastic modulus K, which is a measure of the tissue's elastic stiffness, was assessed in a ~150 µm by 250 µm raster at 20 µm resolution every ~35 minutes, producing a sequence of 'stiffness maps' of the area (Supplementary Fig. 2). To reduce noise, raw . CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The 17, 2018; AFM data were interpolated and smoothed in x-, y-, and time dimensions using an algorithm based on the discrete cosine transform (Fig. 1d, see online methods for details)...

Predicting local tissue mechanics using immunohistochemistry

“…The density of cell nuclei has been found to positively corelate with tissue stiffness in mouse spinal cord tissue (6), ruminant retinae (27), and in Xenopus brains (1, 28). Furthermore, enhancing neurogenesis and thus CNS cell density increased brain tissue stiffness (29), while loss of CNS cells during physiological aging or Alzheimer’s disease both correlated with reduced tissue stiffness (30, 31).…”

“…Furthermore, enhancing neurogenesis and thus CNS cell density increased brain tissue stiffness (29), while loss of CNS cells during physiological aging or Alzheimer’s disease both correlated with reduced tissue stiffness (30, 31). Also, blocking cell proliferation in Xenopus embryos reduced cell densities and tissue stiffness if compared to control brains (28). Together, these studies strongly suggest that the density of cell nuclei directly regulates tissue stiffness.…”