Open Access Highly Accessed Methodology article

Live imaging and analysis of postnatal mouse retinal development

Philip EB Nickerson15, Kara M Ronellenfitch1, Nicklaus F Csuzdi2, Jamie D Boyd3, Perry L Howard14, Kerry R Delaney1 and Robert L Chow1*

Author Affiliations

1 Department of Biology, University of Victoria, Station CSC, PO Box 3020, Victoria, BC V8W 3N5, Canada

2 FRAN, University of Guelph, Guelph, ON, Canada

3 Department of Psychiatry, University of British Columbia, Vancouver, BC, Canada

4 Department of Biochemistry/Microbiology, University of Victoria, Victoria, BC, Canada

5 Current address: Department of Surgery (Neurosurgery), Dalhousie University, Halifax, NS B3H 3A7, Canada

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BMC Developmental Biology 2013, 13:24  doi:10.1186/1471-213X-13-24

Published: 10 June 2013

Abstract

Background

The explanted, developing rodent retina provides an efficient and accessible preparation for use in gene transfer and pharmacological experimentation. Many of the features of normal development are retained in the explanted retina, including retinal progenitor cell proliferation, heterochronic cell production, interkinetic nuclear migration, and connectivity. To date, live imaging in the developing retina has been reported in non-mammalian and mammalian whole-mount samples. An integrated approach to rodent retinal culture/transfection, live imaging, cell tracking, and analysis in structurally intact explants greatly improves our ability to assess the kinetics of cell production.

Results

In this report, we describe the assembly and maintenance of an in vitro, CO2-independent, live mouse retinal preparation that is accessible by both upright and inverted, 2-photon or confocal microscopes. The optics of this preparation permit high-quality and multi-channel imaging of retinal cells expressing fluorescent reporters for up to 48h. Tracking of interkinetic nuclear migration within individual cells, and changes in retinal progenitor cell morphology are described. Follow-up, hierarchical cluster screening revealed that several different dependent variable measures can be used to identify and group movement kinetics in experimental and control samples.

Conclusions

Collectively, these methods provide a robust approach to assay multiple features of rodent retinal development using live imaging.

Keywords:
Postnatal retina; in vitro electroporation; ex vivo culture; Live 2-photon microscopy; Hierarchical cluster analysis