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Disaggregation and Reaggregation of Zebrafish Retinal Cells for the Analysis of Neuronal Layering
The reaggregation of dissociated cells to form organotypic structures provides an in vitro system for the analysis of the cellular interactions and molecular mechanisms involved in the formation of tissue architecture. The retina, an outgrowth of the forebrain, is a precisely layered neural tissue, yet the mechanisms underlying layer formation are largely unexplored. Here we describe the protocol to dissociate, re-aggregate, and culture zebrafish retinal cells from a transgenic, Spectrum of Fates, line where all main cell types are labelled with a combination of fluorescent proteins driven by fate-specific promoters. These cells re-aggregate and self-organize in just 48 h in minimal culture conditions. We also describe how the patterning in these aggregates can be analyzed using isocontour profiling to compare whether different conditions affect their self-organization.
Keywords:3D Petri dish Aggregation Isocontour profiling Müller Glia Organoid Retina Self-organization SoFa Zebrafish
We are grateful to Alexandra D. Almeida for useful discussions during the preparation of this manuscript. We also thank Mark Charlton-Perkins, Ryan MacDonald, and Afnan Azizi for their discussions and contributions to the original research manuscript where the protocols were developed.
This work was funded by a Wellcome Trust Senior Investigator Award to W.A.H. (100329/Z/12/Z) and a Biotechnology and Biological Sciences Research Council.
Studentship Award to M.K.E. (BB/J014540/1).
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