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Fig. 2
A self-organising retina: identification of zebrafish retinal cells and characterisation of organisation. The main cell types of the retina can be identified in the SoFa1 transgenic line (Almeida et al., 2014) using a combination of genetically tagged cell fate markers: Atoh7:gapRFP labels RGC, AC/HC and PR cell membranes; Ptf1a:cytGFP labels AC/HC cytoplasm; and Crx:gapCFP labels BP and PR membranes. (A) Central sagittal section of a region of the SoFa1 retina. Scale bar: 20 μm. (B) Dissociated cells of the SoFa1 line. Scale bar: 20 μm. (C-F) Individual cells are identified based on their spectral expression: (C) RGCs express membrane RFP; (D) AC/HCs express cytoplasmic GFP and membrane RFP; (E) BPs express membrane CFP; and (F) PRs express membrane CFP and RFP. Scale bar: 5 μm. (G-L) Central sagittal section of a retinal aggregate cultured using the SoFa1 line. (G) Crx:gapCFP-expressing cells are found in the centre of the aggregate. (H) Ptf1a:cytGFP-expressing cells are found in a distinct ring around the Crx:gapCFP population. (I) Atoh7:gapRFP-expressing cells are found throughout the aggregate. (J) Merge of channels represented in G-I. (K) DAPI. (L) Bright-field image. Scale bar: 10 μm. (M-P) Generation of analysis of cellular organisation using custom-made Matlab scripts. (M) A mask is fitted to the aggregate using the DAPI channel. (N) Successive isocontours are fitted from the periphery to the centre of the aggregate. (O) Fluorescence is measured along each contour and plotted as a relative fluorescence intensity (y-axis) against radial position (in pixels) (x-axis). (P) Fluorescence profiles for each channel are plotted as an empirical cumulative distribution function (ECDF) (y-axis) against radial position [radial units (ru)] (x-axis). The dotted diagonal line represents a theoretically perfect even distribution of fluorescence from centre to periphery.