Fig. 6. Notch1^- RPCs differentiate predominantly to cone photoreceptors. In the P15 retina of Notch1^f/+;-Cre;Z/AP (A-C,G-I) and Notch1^f/f;-Cre;Z/AP (D-F,J-L) mice, the expression of factors specific for photoreceptor cell types were analyzed using specific antibodies for the detection of recoverin (photoreceptors, B,E) and rhodopsin (rods, C,F), or by labeling with PNA for the detection of cone photoreceptors (C,F) and by detection of transcript using in situ hybridization for Arr3 (cones, H,K) and Gnat1 (rods, I,L). (A-F) Nuclei were visualized by DAPI counterstaining. The distribution of these markers was correlated with AP⁺ activity (A,D,G,J) monitored on adjacent sections (A is adjacent to B,C; D is adjacent to E,F; G is adjacent to H,I and J is adjacent to K,L). In the retina of Notch1^f/+;-Cre;Z/AP, recoverin labels the onl (B), while in the Notch1^f/f;-Cre;Z/AP peripheral retina, the laminar organization is lost and most of the cells are recoverin⁺ (E), demonstrating their differentiation into photoreceptors. Next, analysis of the distribution of cone and rod photoreceptors was performed in correlation with AP distribution. In the normal retina, cones were detected in a few cells in the outer nuclear layer (PNA in C and Arr3 in H), while rods are detected in most of the photoreceptor layer of the control Notch1^f/+;-Cre;Z/AP retina (rhodopsin in C and Gnat1 in I). In the peripheral retina of the Notch1^f/f;-Cre;Z/AP mice, the regions that are AP⁺ (indicated by a broken white line) are mostly populated by PNA⁺;Arr3⁺ cells (F,K), while the AP^- regions (G,J) are occupied mostly by rod photoreceptors (F,L). (M) Quantitative analysis of the ratios of recoverin, Arr3 and Gnat1 to DAPI⁺ areas was conducted on the AP⁺ regions (indicated by the white line; example shown in J-L) detected in the peripheral optic cup. This analysis revealed significant (^*P<0.005) increase in the number of photoreceptors (recoverin⁺) in the Notch1^- retina from 56.6% in the Notch1^f/+;-Cre;Z/AP (s.d.=2.63%, n=3) to 97% (s.d.=1.63%, n=3) in the Notch1^f/f;-Cre;Z/AP mice. Dramatic increase (^*P<0.005) in the number of cone-photoreceptors (Arr3) was detected in the Notch1^- retina from 2% (s.d.=0.2%, n=3) in the Notch1^f/+;-Cre;Z/AP to 89% (s.d.=7.6%, n=3) in the Notch1^f/f;-Cre;Z/AP mice. The number of rods was significantly reduced (^*P<0.005) in the Notch1^- retina from 66.8% (s.d.=2.56%, n=3) in the Notch1^f/+;-Cre;Z/AP to 10.1% (s.d.=4.81%, n=3) in the Notch1^f/f;-Cre;Z/AP mice. gcl, ganglion cell layer; inl, inner nuclear layer; le, lens; onl, outer nuclear layer. Scale bar: 100 µm.