The mito::mKate2 mouse: A far-red fluorescent reporter mouse line for tracking mitochondrial dynamics in vivo.

Mitochondria are incredibly dynamic organelles that undergo continuous fission and fusion events to control morphology, which profoundly impacts cell physiology including cell cycle progression. This is highlighted by the fact that most major human neurodegenerative diseases are due to specific disruptions in mitochondrial fission or fusion machinery and null alleles of these genes result in embryonic lethality. To gain a better understanding of the pathophysiology of such disorders, tools for the in vivo assessment of mitochondrial dynamics are required. It would be particularly advantageous to simultaneously image mitochondrial fission-fusion coincident with cell cycle progression. To that end, we have generated a new transgenic reporter mouse, called mito::mKate2 that ubiquitously expresses a mitochondria localized far-red mKate2 fluorescent protein. Here we show that mito::mKate2 mice are viable and fertile and that mKate2 fluorescence can be spectrally separated from the previously developed Fucci cell cycle reporters. By crossing mito::mKate2 mice to the ROSA26R-mTmG dual fluorescent Cre reporter line, we also demonstrate the potential utility of mito::mKate2 for genetic mosaic analysis of mitochondrial phenotypes.

Live Imaging of Mouse Retinal Slices.

Live fluorescent microscopy of whole-mount rodent retinal explants has proved to be extremely valuable for understanding dynamic events during retinogenesis. However, to obtain three-dimensional images with high-quality axial resolution, investigators are restricted to specific areas of the retina and require microscopes, such as two photon, with a higher level of depth penetrance. As an alternative, we report a retinal live-imaging protocol using slice cultures that are suitable for capturing discrete cellular events during retinal development and differentiation. This is a significant improvement upon current methods, as it is more amenable to a wider array of imaging systems and does not compromise resolution of retinal cross-sectional area.

Live imaging of developing mouse retinal slices.

BACKGROUND: Ex vivo, whole-mount explant culture of the rodent retina has proved to be a valuable approach for studying retinal development. In a limited number of recent studies, this method has been coupled to live fluorescent microscopy with the goal of directly observing dynamic cellular events. However, retinal tissue thickness imposes significant technical limitations. To obtain 3-dimensional images with high quality axial resolution, investigators are restricted to specific areas of the retina and require microscopes, such as 2-photon, with a higher level of depth penetrance. Here, we report a retinal live imaging method that is more amenable to a wider array of imaging systems and does not compromise resolution of retinal cross-sectional area. RESULTS: Mouse retinal slice cultures were prepared and standard, inverted confocal microscopy was used to generate movies with high quality resolution of retinal cross-sections. To illustrate the ability of this method to capture discrete, physiologically relevant events during retinal development, we imaged the dynamics of the Fucci cell cycle reporter in both wild type and Cyclin D1 mutant retinal progenitor cells (RPCs) undergoing interkinetic nuclear migration (INM). Like previously reported for the zebrafish, mouse RPCs in G1 phase migrated stochastically and exhibited overall basal drift during development. In contrast, mouse RPCs in G2 phase displayed directed, apical migration toward the ventricular zone prior to mitosis. We also determined that Cyclin D1 knockout RPCs in G2 exhibited a slower apical velocity as compared to wild type. These data are consistent with previous IdU/BrdU window labeling experiments on Cyclin D1 knockout RPCs indicating an elongated cell cycle. Finally, to illustrate the ability to monitor retinal neuron differentiation, we imaged early postnatal horizontal cells (HCs). Time lapse movies uncovered specific HC neurite dynamics consistent with previously published data showing an instructive role for transient vertical neurites in HC mosaic formation. CONCLUSIONS: We have detailed a straightforward method to image mouse retinal slice culture preparations that, due to its relative ease, extends live retinal imaging capabilities to a more diverse group of scientists. We have also shown that, by using a slice technique, we can achieve excellent lateral resolution, which is advantageous for capturing intracellular dynamics and overall cell movements during retinal development and differentiation.