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Gross CMZ development and anatomy unimpacted by Sema3fa loss. A-D) Retinal sections of WM ISH for vsx2 that becomes restricted to the proliferative retinal periphery in WT (A) and sema3faca304 (B) fish at 52 hpf. At 72 hpf (C-D), vsx2 is expressed at high levels by distal progenitors of the CMZ in both genotypes, but is also expressed at low levels broadly through the mutant nasal CMZ (arrows). Note normal expression of vsx2 by INL cells in both genotypes. n’s represent numbers of fish with represented expression pattern. E-H) Quantitation of the vsx2 expression domain within the CMZ. In sections through the central retina, the vsx2 ISH expression domain and CMZ were outlined, using the edge of the inner and outer plexiform layers to mark the central limit of the CMZ; average CMZ area (E), total vsx2 area normalized to CMZ area (F), and high vsx2 expression area normalized to CMZ area (G, H). I-J) Hoechst labelling of nuclei in horizontal cryostat retinal sections of 7 dpf WT (I) and sema3faca304 (J) nasal (I, J) and temporal (I′, J′) CMZ. K-O) Quantitative analysis of WT and sema3faca304 CMZ at 7 dpf from horizontal sections; (K) CMZ area; (L) Number of Hoechst-labelled nuclei; (O) Average distance between nearest neighbours, represented in M and N. INL, inner nuclear layer; IPL, inner plexiform layer; nr, neural retina; L, lens; RPE, retinal pigment epithelium. For all graphs, data is from N = 3 independent replicates, error bars are standard deviation, values represent individual embryos, and statistics represent Mann Whitney U-test. Scale bar in A is 150 µm for A, B and 40 µm for C, D, I-J, M, N.
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