In vivo imaging of newt lens regeneration: Novel molecular, cellular and functional insights

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Project Summary Regenerating a human organ as its original one remains a part of our imagination. However, newts have unique capabilities of regenerating most of their tissues and organs, even into adulthood, including the lens. If the newt lens is lost or injured, it regenerates from the dorsal iris. Lens regeneration has important clinical significance, but it is also an ideal process for studying tissue regeneration in general. Many ex-vivo technologies, such as histological analysis, can only show us a snapshot of the regeneration process from a specific point of view at a specific time point. However, lens regeneration is a dynamic process involving cellular, molecular and functional changes. We have been able to, for the first time, non-invasively acquire high-quality in vivo images during the process of lens regeneration with optical coherence tomography (OCT) by tracking a single newt for over a period of 40 days. More interestingly, OCT was able to image the fragile zonular fibers for the first time during this process. This has not been documented using histological/immunohistological analysis. In addition, the blood vessels in the iris stroma are also clearly visible. In this proposal, we will significantly advance the imaging technology currently available. We hypothesize that by integrating high-resolution OCT and confocal fluorescence laser scanning microscopy (CFLSM), in combination with the use of newts lacking pigments in the iris and lineage tracing transgenic newts, we will be able to in vivo image the molecular, cellular, and functional changes taking place during the process of lens regeneration. To achieve this, we will custom-build a multimodality imaging system with high resolution, sufficient imaging depth, and functional imaging capabilities. In the three aims proposed, we will reveal the detail process of lens regeneration, including lens vesicle formation, lens fiber differentiation, the development of the zonular fibers, the changes in iris vasculature and the dynamic distribution of macrophages using a combination of mutant and transgenic newts. After completing these aims, we will have established a new comprehensive imaging platform, that will allow researchers to in vivo track the process of lens regeneration in a single newt without interruptions and contribute critical information that can be used to understand cataract biology, zonulopathies and lens replacement where intact lens capsules are absent in humans