Abstract
ABSTRACTTo investigate the cell-cell interactions necessary for the formation of retinal layers, we cultured dissociated zebrafish retinal progenitors in agarose microwells. Within these wells, the cells re-aggregated within hours, forming tight retinal organoids. Using a Spectrum of Fates zebrafish line, in which all different types of retinal neurons show distinct fluorescent spectra, we found that by 48 h in culture, the retinal organoids acquire a distinct spatial organisation, i.e. they became coarsely but clearly laminated. Retinal pigment epithelium cells were in the centre, photoreceptors and bipolar cells were next most central and amacrine cells and retinal ganglion cells were on the outside. Image analysis allowed us to derive quantitative measures of lamination, which we then used to find that Müller glia, but not RPE cells, are essential for this process.
Highlights
The retina is a strikingly well-organised neural tissue, with each of the major cell types sitting in its own specific layer
Dissection, dissociation and culture of zebrafish retinal cells At 24 h post fertilisation, the zebrafish retina is a pseudostratified epithelium comprising ∼2000 progenitor cells, each stretching from the apical to the basal surface
After disaggregation, dissociated zebrafish retinal progenitors reaggregate quickly; within only 48 h in culture, they are able to organise themselves into layers
Summary
The retina is a strikingly well-organised neural tissue, with each of the major cell types sitting in its own specific layer. Such laminated cellular organisation, which is common in the nervous system, may aid in wiring the brain efficiently during development. Timing alone does not account for this organisation, as is clearly shown in the example of reeler mutant mice, where the neocortex, shows the opposite ‘outside-in’ order of histogenesis, even though the different types of cortical cells are generated and migrate to the cortical plate at the correct times (Caviness and Sidman, 1973). The layering defect in reeler is due to the lack of the glycoprotein reelin, which is secreted largely by a single transient cell type, the Cajal-Retzius cell (D’Arcangelo and Curran, 1998; Huang, 2009), suggesting certain cells and molecules play important roles in histogenesis
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