Fig 5

Disrupted organization of committed CMZ progenitors in the absence of Sema3fa.

WM ISH for Zone 3 markers shown in 7 µm plastic sections (A, B, E, F, I′, J′, K, L), z-stack of confocal optical sections (M, N), or lateral WM (I, J). A-H)cdkn1c (A, B) and atoh7(E, F) ISH at 72 hpf, graphs of cdkn1c(C) (pooled data from N = 2 independent replicates) and atoh7(G) (pooled data from N = 4 independent replicates) mRNA domain areas normalized to CMZ area; Points for graphs are independent biological replicates, errors are standard deviation, and statistics are Mann Whitney U-test. Fold change of cdkn1c (D) and atoh7 (H) mRNA levels in 72 hpf eyes relative to WT as measured by RT-qPCR (paired Student’s t-test of delta CT values, with each data point an average of 3 technical replicates of RT-qPCR performed on mRNA isolated from n = 30 eyes). I-J) Zone 3 hes6 marker in the 72 hpf eye in WM (I, J) and in plastic section (I′, J′). Expansion of hes6 in situ label to the most peripheral CMZ adjacent to the lens in sema3fa^ca304 eye (yellow arrows). n’s refer to the numbers of eyes with significant (J) or little or no (I) hes6 ISH label in the CMZ immediately adjacent to the lens. K, L) Plastic section of Zone 3 neurod4 mRNA in the 72 hpf CMZ showing expansion (yellow arrows) of the neurod4 label towards peripheral CMZ. M, N) Confocal images of 72 hpf CMZ processed for WM double FISH for ccnd1 (red) and neurod4 (green). There is more co-expression in mutant than WT (compare arrows in M and N). n’s refer to the numbers of larvae showing WT levels of co-expression (M) and enhanced co-localization (N). O, P) Double FISH for ccnd1 (O, P) and atoh7 (O′, P′), and merge (O′′, P′′; green-ccnd1, red-atoh7) for WT (O) and mutant (P) 72 hpf CMZ. Q-R) Quantitation of Hoescht-labelled nuclei in ccnd1+ (Q) and atoh7+ (R) expression domains in transverse retinal sections processed for double FISH for ccnd1 and atoh7. Points for graphs are individual larva, errors are standard deviation, and statistics are Mann Whitney U-test (N = 2 independent experiments). Scale bar in A is 20 µm for all panels except I, J (100 µm).