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, 13 (1), 23

Live Imaging of Developing Mouse Retinal Slices

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Live Imaging of Developing Mouse Retinal Slices

Anthony P Barrasso et al. Neural Dev.

Abstract

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.

Keywords: Cyclin D1; Horizontal neurons; Interkinetic nuclear migration; Live imaging; Mouse retinal progenitor cells.

Conflict of interest statement

Ethics approval and consent to participate

All animal research was conducted according to protocols approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Schematic of retinal slice culture protocol
Fig. 2
Fig. 2
Analysis of retinal slice survival and proliferation. Retinal slice cultures stained with Zombie Red™ dye after 0 h (a and c) and 16 h (b and d) in culture. Higher magnifications of boxed region in A-D (E-H). The quantification of Zombie Red+ cells (ZR+ pixels/retinal area) showed no significant differences between 0 h and 16 h in culture (i) or between tissue exposed to laser versus unexposed (j). n = 9 per group. EdU labeling (6-h pulse) and quantification of RPCs in P0 and P1 retinae compared to P0 retinal slices time lapse cultures (k-n). n = 3 per group. Error bars represent SE. Abbreviations: NBL (neuroblastic layer), IPL (inner plexiform layer), GCL (ganglion cell layer)
Fig. 3
Fig. 3
Schematic of two models of INM. The “elevator” model (a) and the “stochastic” model (b). See text for details
Fig. 4
Fig. 4
Characterization of Fucci+ cells in the P0 retina. Fucci expression throughout the cell cycle (a). P0 Fucci retinae labelled with anti-AzG (b), MCM6 (c), PH3 (d), and Calbindin (e). n > 3
Fig. 5
Fig. 5
Tracking INM in the P0 retinae. Stills from time lapse movie of migrating RPC nuclei. Arrows indicate apically migrating AzG+ (G2-phase) nuclei (a). Relative position of nuclei over time (b-c). MSD of nuclei plotted over time (d-e). Comparison of the average apical velocity of AzG+ cells over the course of the time lapse experiments (f)
Fig. 6
Fig. 6
Apical migration in the Cyclin D1−/− retina. Still images from time lapse movies of migrating wild type and mutant AzG+ RPCs and the overall displacement of each nucleus (a-b). Relative position of nuclei over time (c-d). The distributions of average and maximum apical velocities of tracked nuclei (e-f). Data were collected from 56 nuclei in 3 WT retinae and 54 nuclei in 3 KO retinae. ***p < 0.001
Fig. 7
Fig. 7
Live imaging of horizontal cells. Still image from a time lapse movie of a P2 Cx57-iCre+/tg; Rosa26R-mTmG+/tg retina (a). Center of mass distance measurement indicating little HC somal translocation (b). Selected images series from a time lapse movie of two horizontal cells (c). The colored arrows indicate dynamic, vertically-oriented neurites that do not overlap with neurites of the adjacent cell. The white arrowheads indicate more laterally-oriented neurites that exhibit overlap with the adjacent cell. Tracings of HC neurites with selected vertical neurite traces colored (d). Highlighted territories of overlap between adjacent HCs (e). Measurements of total HC area (f) and neurite overlap over time (g). n > 3 movies

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