Notch/Hes signaling pathway is activated in the add mutant retina. (A) Lateral view of 34 hpf wild-type retinas expressing ΔN-Tcf3. A confocal image of a 4.5 µm plane is shown. Although ath5:GFP expression (green) progresses to the large region of the neural retina in this stage (see Fig. 1D), ath5:GFP spreads to the dorsonasal retina in this plane. Some of ΔN-Tcf3-expressing cells (red) are ath5:GFP positive (arrows, yellow). (B) A confocal image of lateral view of 34 hpf add mutant retinas expressing ΔN-Tcf3. Very faint ath5:GFP expression is detected in the ventronasal retina (green, arrowhead). Cells expressing ΔN-Tcf3 (red) in the dorsal retina do not express ath5:GFP (arrows). (C-D) In situ hybridization of wild-type (C) and add mutant (D) retinas with her4 RNA probe. In the wild-type retina, her4 expression is localized in the central part of the CMZ (lines and arrows), which corresponds to the intermediate zone between retinal stem cells (asterisks) and differentiated neurons. her4 expression is also observed in a part of the outer layer, where neurogenesis occurs at this stage. By contrast, her4 expression remains in the large region of the neural retina in the add mutant (D). (E,F) In situ hybridization of wild-type (E) and wild-type retinas expressing NICD (F) with her4 RNA probe. her4 expression is upregulated in NICD-expressing retinas. (G,H) In situ hybridization of mib (G) and add; mib double mutant (H) retinas with her4 RNA probe. her4 expression is not observed in either case. (I,J) In situ hybridization of wild-type (I) and wild-type retinas expressing Δ47-ß-catenin (J) with her4 RNA probe. her4 is expressed in the CMZ and the presumptive outer layer, the latter of which is undulated because of Δ47-ß-catenin-induced folding of the neural retina.
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