Live imaging of developing mouse retinal slices

Abstract

BackgroundEx 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.ResultsMouse 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.ConclusionsWe 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.Electronic supplementary materialThe online version of this article (10.1186/s13064-018-0120-y) contains supplementary material, which is available to authorized users.

Nuclear crowding and nonlinear diffusion during interkinetic nuclear migration in the zebrafish retina

“…Most daughter nuclei move away from the apical surface, within minutes of being born, with a clear basalward bias in their speed distribution ( Figure 2C ). This abrupt basal motion of newly divided nuclei has also been recently observed by others ( Leung et al, 2011 ; Shinoda et al, 2018 ; Barrasso et al, 2018 ). However, immediately after this brief period, nuclear speeds become much more equally distributed between basalward and apicalward, with a mean value near 0.…”

The Hippo Pathway Blocks Mammalian Retinal Müller Glial Cell Reprogramming

“…Slides were incubated for 24 hours in primary antibodies in a humid chamber. The following antibodies and concentrations were used: mouse-YAP (1:100, Santa Cruz, #101199,( Szymaniak et al, 2017 ; Zanconato et al, 2015 )), rabbit-pYAP (1:500, Cell Signaling, #4911, ( Heallen et al, 2013 )), rabbit-SOX9 (1:500, Millipore, AB5535,( Poché et al, 2008 )), mouse-TEAD1 (1:1000, BD transduction, 610923, ( Cao et al, 2008 )), mouse-SOX2 (1:500, Santa Cruz, 365823, ( Xiao et al, 2018a )), rabbit-GFAP (1:1000, Dako, Z0334, ( Poché et al, 2016 )), rabbit-PH3 (1:500, Millipore, 06–570) ( Barrasso et al, 2018 ), rabbit-Ki67 (1:500, Abcam 15580, ( Poché et al, 2016 )), rabbit-Cyclin D1 (1:1000, Thermo Fisher, 9104, ( Poché et al, 2016 )), mouse-CYCLIN D3 (1:200, Abcam, 28283, ( Wang et al, 2017 )), rabbit-GFP (1:500, Rockland, 600–401-215, ( Kovalchuk et al, 2015 )), mouse-Flag (1:250, Sigma, F1804, ( Zanconato et al, 2015 )), chick-beta-Gal (1:500, Abcam, 936, ( Kim et al, 2018 )), mouse-GS (1:500, Chemicon, MAB302, ( Rueda et al, 2016 )), mouse-HuC/D (1:100, Invitrogen, A-21271, ( Jorstad et al, 2017 ; Ueki et al, 2015 )).…”

Photoreceptor degeneration in microphthalmia (Mitf) mice: partial rescue by pigment epithelium-derived factor

“…Intact retina and lens were exposed and isolated by gently tearing the sclera at the optic nerve foramen. For retina explant culture, explants were placed in a drop of medium (100 µl) on a culture nucleopore track-etched polycarbonate membrane (Whatman, 110410) floating in a 35-mm dish filled with 2 ml control culture medium [CM; 45% Dulbecco's modified Eagle medium (DMEM; Invitrogen, 12430), 45% DMEM/F12 (Invitrogen, 11330), 10% fetal bovine serum, 1× Insulin-Transferrin-Selenium (Invitrogen), and 1× HEPES, a modification of the retina explant medium described by Barrasso et al (2018)] or CM supplemented with PEDF or the 17-mer peptide at different dosages as described in the Results. For recombination experiments, eyeballs from P8 WT and Mitf −/− mice were harvested and the scleras were torn open as mentioned to obtain the neural retina.…”