Eph receptors and their membrane-bound ligands, ephrins, provide key signals in many developmental processes including axon guidance. In the nervous system, concentration gradients of Ephs and ephrins are crucial in guiding retinal ganglion cell (RGC) axons from the retina to the right targets in the visual area of the brain, the optic tectum. However, despite immense progress in our understanding of Eph/ephrin signalling in neural development, discrepancies between in vitro and in vivo work remain. As axon pathfinding is regulated by chemical and mechanical signals, and the mechanical regulation of Eph/ephrin signalling is currently poorly understood, I investigated the role of tissue stiffness in this signalling pathway using the optic pathway of the African clawed frog, Xenopus laevis, as a model system.

Using atomic force microscopy, I found that, in vivo at early stages, the Xenopus optic tectum was soft and mechanically homogenous. However, as RGC axons approached the optic tectum, a stiffness gradient developed across it, with low stiffness anteriorly to high stiffness posteriorly. This stiffness gradient correlated both spatially and temporally with the emergence of a nuclear density gradient, suggesting that local cell density changes may contribute to the increase in stiffness in the posterior tectum.

Subsequently, I investigated EphB mRNA levels in the optic tectum at a time point when RGC axons are approaching and innervating the optic tectum. I found EphB1 mRNA levels to be higher in the anterior than posterior tectum. This pattern of mRNA expression emerged at similar developmental stages as the cell density and stiffness gradients did, indicating a negative correlation between EphB mRNA expression and tissue stiffness.

As RGC axons growing through the optic tectum express different levels of EphB, I next investigated how substrate stiffness affects this signalling family in vitro. In agreement with previous studies performed on glass and tissue-culture plastics, ventral RGCs responded to ephrinB on glass, while dorsal RGCs did not. However, on soft substrates mechanically mimicking brain tissue, both dorsal and ventral RGCs responded to ephrinB to the same degree. This suggested that on substrates of stiffness similar to that of the developing optic tectum in vivo, EphB forward signalling does not differentially affect dorsal and ventral RGCs. To investigate the underlying mechanosensory component of EphB forward signalling, I optimised assays investigating EphB mRNA and protein localisation, as well as endocytosis. I validated my findings on glass using immunostainings, showing that ventral RGC growth cones express higher EphB levels than dorsal RGC growth cones.

Concurrently, I investigated the role of tissue stiffness in RGC axon unbundling as the optic tract innervates the optic tectum. I characterised the mechanical landscape in the vicinity of the optic tract and found a stiffness gradient across the diencephalon-tectum boundary at the time of innervation by RGC axons. To characterise how substrate stiffness affects axon fasciculation independently of chemical cues, I developed an in vitro assay combining stiffness gradient hydrogels and micropatterning.

Since Eph/ephrin signalling in Xenopus RGCs is affected by substrate stiffness in vitro, and a stiffness gradient develops across the optic tectum at the time of innervation, my results suggest that mechanical cues could be important in tuning axon guidance through the regulation of chemical signalling. A similar regulation of Eph/ephrin signalling through tissue mechanics is likely to be important across multiple aspects of neural development, as well as in other organ systems.