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Fig. 6. Notch1- RPCs differentiate predominantly to cone
photoreceptors. In the P15 retina of
Notch1f/+;
-Cre;Z/AP
(A-C,G-I) and
Notch1f/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
Notch1f/+;-Cre;Z/AP, recoverin labels the
onl (B), while in the Notch1f/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 Notch1f/+;-Cre;Z/AP
retina (rhodopsin in C and Gnat1 in I). In the peripheral retina of the
Notch1f/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
Notch1f/+;-Cre;Z/AP (s.d.=2.63%,
n=3) to 97% (s.d.=1.63%, n=3) in the
Notch1f/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
Notch1f/+;-Cre;Z/AP to 89% (s.d.=7.6%,
n=3) in the Notch1f/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
Notch1f/+;-Cre;Z/AP to 10.1% (s.d.=4.81%,
n=3) in the Notch1f/f;-Cre;Z/AP
mice. gcl, ganglion cell layer; inl, inner nuclear layer; le, lens; onl, outer
nuclear layer. Scale bar: 100 µm.
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