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Figure 4


Fig. 4. Reduction in the mitotic index and the number of ganglion and horizontal cell types in the Notch1- retina. (A-C) Fewer BrdU+ nuclei (BrdU+ in red, DAPI in blue; A,B) are detected in the Notch1f/f;{alpha}Cre (B) when compared with the control Notch1f/+;{alpha}-Cre (A) retina. (C) The proportion of BrdU+/DAPI+ area was 32.7% in the Notch1f/+;{alpha}-Cre eyes (s.d.=2 %, n=4), whereas only 25.8% BrdU+/DAPI+ area was detected in the Notch1f/f;{alpha}-Cre retina (s.d.=3.4%, n=4). Thus, the mitotic index in the Notch1f/f;{alpha}-Cre retina is significantly reduced, to about 78% of the control (*t-test, P<0.02). (D,E) The ganglion cells (Brn3b, red) and horizontal cells (Nf165, green) are detected in the peripheral retina of E17.5 control Notch1f/+;{alpha}-Cre;Z/AP (D) eyes. Reduction in the number of Brn3b+ and almost complete loss of Nf165+ cells is observed at this stage in the Notch1f/f;{alpha}Cre;Z/AP peripheral retina (E). (F,G) Counterstaining with DAPI (blue) reveals the abnormal retinal morphology and rosettes (white arrowheads, G) in the Notch1- but not in the Notch1+ peripheral retina (F). (H-K) Quantitative evaluation of the reduction in Brn3b+ cells in the Notch1- RPCs was conducted by dissociation of E14 retinas from control Notch1f/+;{alpha}-Cre;Z/AP or mutant Notch1f/f;{alpha}-Cre;Z/AP embryos. The cell nuclei were visualized by counterstaining with DAPI (blue, H). The Brn3b+ cells (red, I) were detected by indirect immunofluorescent analysis and documented. On the same slide, AP activity was monitored (purple, J). The proportion of Brn3+;AP+ was calculated from the total number of AP+ cells (K). A significant reduction of 35% (K, t-test, *P<0.02) in the proportion of Brn3b+;AP+ positive cells from AP+ cells was observed in the Notch1f/f;{alpha}-Cre;Z/AP dissociated retina (average=4.4±2%, n=6 embryos) when compared with the Notch1f/+;{alpha}-Cre;Z/AP control retinas (average=12.9±3.7%, n=6 embryos). gcl, ganglion cell layer; le, lens; nbl, neuroblastic layer. Scale bars: 100 µm.





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