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First published online 1 March 2006
doi: 10.1242/dev.02311

Development 133, 1367-1378 (2006)
Published by The Company of Biologists 2006
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Notch1 functions to suppress cone-photoreceptor fate specification in the developing mouse retina

Orly Yaron1,*, Chen Farhy1,*, Till Marquardt2, Meredithe Applebury3 and Ruth Ashery-Padan1,{dagger}

1 Sackler Faculty of Medicine, Department of Human Molecular Genetics and Biochemistry, Tel Aviv University, Tel Aviv 69978, Israel.
2 The Salk Institute for Biological Studies, Gene Expression Laboratory, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.
3 The Howe Laboratory, Harvard Medical School, Boston, MA 02114, USA.


Figure 1
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  		Fig. 1. Notch1 inactivation in retinal progenitor cells. (A) Notch1 protein includes a signal peptide (SP), 36 epidermal growth factor-like repeats (EGF), three lin repeats (LN), a transmembrane domain (TM), six Cdc10 ankyrin-like repeats, a polyglutamine stretch (opa), and a proline, serine and threonine stretch (PEST) (del Amo et al., 1993Go). The Notch1f allele includes two loxP sites (black arrowheads) around the first exon encoding Notch1 signal peptide (Radtke et al., 1999Go). (B) {alpha}-Cre transgene drives the expression of Cre recombinase and a downstream GFP reporter under a Pax6 {alpha}-enhancer in RPCs of the distal retina (Marquardt et al., 2001Go). (C) The Z/AP transgene construct used for lineage tracing of Cre-expressing cells. Cre-mediated recombination of the Z/AP transgene eliminates the lacZ cassette, enabling expression of the reporter gene human placental alkaline phosphatase (AP). (D-L) Serial sections of E12.5 (D-G) and E13.5 (I-L) {alpha}-Cre;Z/AP control eyes characterized for AP activity (D,I), Notch1 (E,J) and Hes5 (F,K) expression by in situ hybridization, and for Brn3b (G,L) detected by indirect immunofluorescent analysis. (H,M) Summary of the expression patterns along the central (c), peripheral (p) and the non-neuronal tip (t) of the optic cup is presented for E12.5 (H) and E13.5 (M). Notch1 and Hes5 expression already partially overlaps with AP at E12.5, while Brn3b expression seems to overlap with the AP+ domain at E13.5. le, lens; oc, optic cup; gc, ganglion cells. Scale bar: 100 µm.

 

Figure 2
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  		Fig. 2. Microphthalmia and disrupted retinal morphology following Notch1 inactivation in the RPCs. (A) Normal eye size is observed in the control, while microphthalmia is evident in the Notch1f/f;{alpha}-Cre mice (E). (B-D,F-H) H&E staining of eye sections from P15 (B,F), E17.5 (C,G) and E13.5 (D,H) embryos demonstrating the normal retinal morphology in the control eyes (B-D), while in the Notch1f/f;{alpha}-Cre eyes rosettes are detected in the retina (F-H) as early as E13.5 (H). (I) Diagram illustrating the lineage tracing of the Notch1- cells employing the Notch1f/f;{alpha}-Cre;Z/AP mice. In these mice, Notch1 gene remains intact and AP is not expressed in cells that have not expressed Cre. Notch1, however, is deleted and AP activity detected in cells that expressed Cre and in their progeny. (J-Q) AP activity was monitored on sections from control (J-M) and Notch1f/f;{alpha}-Cre;Z/AP (N-Q) eyes. At P15 (J,K) and P0 (L), AP is detected throughout the peripheral {alpha}-Cre;Z/AP retina as expected from the early activity of Cre observed in most RPCs in this region (M). In the Notch1f/f;{alpha}-Cre;Z/AP P15 eyes, however, AP+ (Notch1-) cells are dramatically reduced in number thus most of the peripheral retina is missing (N,O). The iris maintains its normal structure despite loss of retinal tissue (compare J with N). At P0 (P), fewer AP+ cells are detected in the Notch1f/f;{alpha}-Cre;Z/AP retina when compared with the wide distribution of AP+ cells in the control (L). The rosettes at P0 are composed of a mixture of AP+ (Notch1-) and AP- (Notch1+) cells. However, at E13.5 (Q), when rosettes first appear in the Notch1f/f;{alpha}-Cre;Z/AP, the distribution of AP is similar to its distribution in the controls (M), encompassing most of the peripheral retina and the rosettes seem to be mostly composed of AP+ cells (Q, inset). The red arrowheads in J,N,L,P indicate the AP+ amacrine precursors that seem to be unaffected following Notch1 inactivation. Control genotypes: Notch1f/+;{alpha}-Cre (A-D); Notch1f/+;{alpha}-Cre;Z/AP (B-D,J-M). co, cornea; gc, ganglion cells; inl, inner nuclear layer; i, iris; le, lens; nr, neuroretina; onl, outer nuclear layer; rpe, retinal pigmented epithelium. Scale bars: 100 µm in F,H,O,Q; 200 µm in G,P; 500 µm in N.

 

Figure 3
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  		Fig. 3. The expression profile of proneural bHLH genes is altered in the Notch1- retina. To visualize the domain of {alpha}-Cre-mediated recombination, AP activity was monitored on sections from E13.5 {alpha}-Cre;Z/AP control (A) and Notch1f/f;{alpha}-Cre;Z/AP mutants (H). The black arrowheads indicate the peripheral retina where Cre activity is detected. The expression of Hes5 (B,I), Neurod1 (C,J), Ngn2 (D,K), Mash1 (E,L), Math5 (F,M) and Math3 (G,N) were determined by in situ hybridization. In control eyes (B-G), the normal pattern of bHLH gene expression was observed, while a change in the expression profile of these factors was detected in the Notch1f/f;{alpha}-Cre;Z/AP eyes (I-N). The expression of Hes5 was reduced (compare I with B), the expression of Neurod1, Ngn2, Math5 and Math3 was enhanced (compare J,K,M,N with C,D,F,G), and the expression of Mash1 appears unchanged (compare L with E) in the Notch1f/f;{alpha}-Cre;Z/AP when compared with {alpha}-Cre;Z/AP controls. le, lens; oc, optic cup. Scale bar: 100 µm.

 

Figure 4
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  		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.

 

Figure 5
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  		Fig. 5. Notch1- RPC differentiation into precursors of photoreceptors. The expression of factors involved in photoreceptor differentiation Otx2 (A,C,F,H), Crx (B,D,G,I) and Thrß2 (E,J) was analyzed by in situ hybridization on control (A-E) and Notch1f/f;{alpha}-Cre (F-J) eyes. In the control E13.5 (A,B) and E16.5 (C-E) retinas, the expression of Otx2 (A,C), Crx (B,D) and Thrß2 (E) is detected in the outer neuroblastic layer (onb). In the Notch1f/f;{alpha}-Cre retinas, the expression of Otx2 (F,H), Crx (G,I) and Thrß2 (J) is detected in the different layers of the peripheral retina. le, lens; onb, outer neuroblastic layer; RPE, retinal pigmented epithelium. Scale bar: 100 µm.

 

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

 

Figure 7
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  		Fig. 7. A scheme summarizing the possible functions of Notch1 in maintaining the multipotency of retinal progenitor cells and in photoreceptor cell differentiation. During normal retinal development (A), Notch1 is expressed in RPCs (blue) and is required for the expression of Hes1 and Hes5, which function to repress proneural gene expression and thus to inhibit cell differentiation. Release from Notch1 inhibition occurs gradually, in only a few cells at a time, and these cells will differentiate into the different retinal cell types (G, ganglion; C, cone; A, amacrine; H, horizontal; BPL, bipolar; MG, Mueller glia). Inactivation of Notch1 in the RPCs during early stages of retinogenesis does not result in premature differentiation into the different early cell types (B). The premature loss of Notch1 activity resulted in differentiation of most RPCs into cone-photoreceptor precursors expressing Otx2, Crx and the early cone marker Thrß2. These cone precursors differentiate to mature cones during postnatal stages, thus following the normal temporal order of the differentiation of photoreceptors.

 

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© The Company of Biologists Ltd 2006