Fig 7

Sema3fa may promote smooth movement of progenitors through CMZ by downregulating cell adhesion.

A-B) Radial EdU+ cohorts (green) that have moved away (dotted arrows) from the CMZ in WT (A) and sema3fa^ca304 retina (B). A 4 hour EdU pulse was performed at 7 dpf and retinal sections from larvae fixed at 20 dpf processed for EdU labelled cells. C) Average distance (d₁; D) of EdU+ radial cohorts from the CMZ in sema3fa^ca304 is reduced significantly as compared to WT. N = 3 independent replicates. D) Schematic showing measurement of the distance (d₁) moved from the central CMZ edge by radial cohorts of EdU+ cells (green) 2 weeks post-EdU label, and the distance (d₂) moved from the peripheral CMZ edge by the central-most cohort of EdU+ cells (green) 3 days post-EdU label. E-F) Graphs showing the average distance (d₂; D) of each radial cluster of EdU-labelled CMZ-derived cells from the peripheral-most CMZ, as measured in 9 dpf dorsal (E) and ventral (F) retina. N = 2-3 independent replicates. G-H) Retinal sections of the CMZ from WM ISH for plxna3 mRNA in WT (G) and sema3fa^ca304 (H) 72 hpf fish. n’s represent the numbers of larva showing either WT (G) or expanded (H; arrows) plxna3 expression. I-J) WT (I) and sema3fa^ca304(J) CMZ processed by double FISH for plxna3 (green) and ccnd1 (red) mRNA. plxna3-expressing CMZ cells are found ectopically (arrows) in the peripheral CMZ and co-express ccnd1 (yellow). n’s represent the number of individual embryos that exhibit separated (I) or overlapped (J) ccnd1/plxna3 expression domains. K) Quantitation of plxna3 expression domain normalized to CMZ area of 72 hpf horizontal retinal sections (N = 3 independent replicates). L-O) Immunolabel for ß-catenin (L, M) and Cdh2 (N, O) in the 72 hpf CMZ. Arrows indicate reduced immunolabel of the central CMZ in WT but not mutant. n’s indicate the numbers of fish showing either preferential, bright label of the more distal CMZ (L, N) or immunolabel distributed across both the distal and central CMZ (M, O). P) Quantitation of the percentage of the CMZ exhibiting robust Cdh2 immunostaining fluorescent label. Data from N = 4 independent replicates. Q-S) Cdh2 immunolabelling of the CMZ in larvae injected at the 1-cell stage with a hsp70:sema3fa-myc construct and heat-shocked at 38°C for 60 minutes at 52 hpf and fixed 24 hours later. Quantitation (S) of the domain of high Cdh2 expression as a percentage of CMZ area in CMZ exhibiting Myc+ cells (R, R’; red) and no Myc+ cells (Q). Data from N = 3 independent replicates. For all graphs, dots represent individual fish, error is standard deviation, and Mann Whitney U-test was performed for statistical analysis. T) Schematic of the CMZ of WT and sema3fa^ca304 fish showing normal retinal stem cell (RSC) localization to the CMZ periphery, but disrupted organization of committed and proliferating (retinal progenitor cells; RPC) in the mutants. Fewer neurons (purple) are produced and move less distance from the CMZ in mutants. We propose a model whereby Sema3fa negatively regulates cell adhesion molecule expression by progenitors. As such, sema3fa^ca304 CMZ progenitors are more adhesive and do not move smoothly through the CMZ (red cells, arrows). Boxes show impact on the zonal organization of the CMZ. Scale bar in A is 100 µm for A, B and 25 µm for G-J, L-O, Q-R.