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First published online 1 February 2006
doi: 10.1242/dev.02245

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Notch 1 inhibits photoreceptor production in the developing mammalian retina

¹ Department of Genetics and Howard Hughes Medical Institute, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
² Developmental Genetics Program and the Department of Cell Biology, The Skirball Institute of Biomolecular Medicine, New York University Medical Center, New York, NY 10016, USA.

View larger version (75K): [in a new window]   Fig. 1. Morphology of Notch1 CKO retinae. (A,B) Hematoxylin and Eosin staining of retinae from 3-week old wild-type (A) and Notch1 CKO (B) mice. The white arrowheads indicate rosette-like structures. (C) Schematic illustration of NIN and NIN-CRE constructs. Representative two- and one-cell clones from NIN infection. (D) Quantitation of clone sizes resulting from NIN or NIN-CRE infections on Notch1 flox/flox retinae. Two independent retinae totaling 300-350 clones were analyzed for each type of virus. Change in clone size in the NIN-CRE-infected retinae compared with control (NIN-infected retinae) is statistically significant (*P<0.05, two-tailed t-test assuming equal variances). GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, outer segment.

View larger version (100K): [in a new window]   Fig. 2. Fate mapping of Notch1 ablated retinal cells at embryonic timepoints. (A) Schematic illustration of transgenic constructs. The floxed Notch1 allele was generated by flanking the first coding exon with LoxP sequences. Removal of exon1 of Notch1 with cre recombinase removes the exon encoding the signal peptide and leads to the generation of a null allele. ROSA26-R (R26R) is a cre recombinase reporter comprising a LoxP flanked stop codon preceding a ß-galactosidase coding region (lacZ). The Chx10-CRE mice contain a BAC transgene consisting of a cre-GFP fusion knocked into the Chx10 promoter. Notch1-ablated retinae were generated by crossing the Chx10-CRE allele into Notch1 flox/flox mice. Fate mapping of recombined cells was possible by X-gal staining in mice additionally containing the R26R allele. (B-E) Fate mapping of wild-type (B,C) and Notch1 ablated (D,E) retinal progenitor cells at E16.5 (B,D) and E18.5 (C,E), as detected by X-gal staining. INBL, inner neuroblastic layer; ONBL, outer neuroblastic layer.

View larger version (155K): [in a new window]   Fig. 3. Expression of markers of RPCs and enhanced neurogenesis in Notch1 deficient retinae. Section in situ hybridization on wild type (A-D,I-L) and Notch1 CKO (E-H,M-P) retinae at E13.5. (A,E) Notch1, (B,F) Hey1, (C,G) cyclin D1, (D,H) Fgf15, (I,M) Dll1, (J,N) Hes6, (K,O) Neurod1, (L,P) Math3.

View larger version (35K): [in a new window]   Fig. 4. Gene changes in Notch1 ablated retinae at E13.5. Retinae from wild-type and Notch1 CKO littermates were harvested at E14.5, pulsed with BrdU in vitro for 1 hour and dissociated. (A) DAPI-positive cells were scored for immunoreactivity for ß-tubulin III, 270.7 or BrdU. Two-thousand to 3000 cells were scored from two or three independent retinae for each genotype. Change in immunoreactive cells in the Notch1 CKO retinae compared with control (wild type) is statistically significant (*P<0.05, two-tailed t-test assuming equal variances). Retinae from four wild-type and Notch1 CKO littermates were harvested at E13.5 and processed for RNA isolation and cDNA preparation. cDNA was amplified, labeled and hybridized to cDNA microarrays. (B) Scatterplot representing the ratio of gene expression in wild-type versus mutant samples (y axis) plotted against the intensity value in the mutant sample (x axis). Each spot corresponds to one gene. Any spot that lies above 1 on the y axis represents a gene that is expressed at a higher level in the wild-type tissue; conversely, any spot that lies below 1 on the x axis represents a gene that is higher in the Notch1 CKO tissue. (C,D) Selected genes expressed at a lower (C) or higher (D) level in the Notch1 CKO are summarized with gene name, Accession Number, average fold change and s.d. in a dye swap experiment. *Some spots on array that correspond to laboratory clones, sequences are available upon request. The complete set of microarray results are included in Data S1 in the supplementary material.

View larger version (162K): [in a new window]   Fig. 5. Gene expression in Notch1 ablated retinae at E13.5. Gene changes were examined by section in situ hybridization on wild-type (A-D), Notch1 flox/flox; Chx10-CRE (E-H), Notch1 flox/flox (I-L), and Notch1 flox/flox; Foxg1-CRE (M-P) retinae at E13.5. (A,E) Otx2, (B,F,J,N) Crx, (C,G,K,O) Nefl, (D,H,L,P) islet 1, (I,M) cyclin D1.

View larger version (113K): [in a new window]   Fig. 6. Fate mapping and gene expression in mature Notch1 ablated retinae. Fate mapping of wild-type (A,D) and Notch1-ablated (B,C,E,F) retinal progenitor cells at P14 as detected by X-gal staining at high (A-C) and low (D-F) magnification. INL, inner nuclear layer; ONL, outer nuclear layer; OS, outer segment.

View larger version (125K): [in a new window]   Fig. 7. Gene expression in mature Notch1 ablated retinae. Section in situ hybridization on wild-type (A-E,K-O) and Notch1 CKO (F-J,P-T) retinae at P15. (A,F) M-Opsin, (B,G) S-Opsin, (C,H) Gap43, (D,I) Nefl, (E,J) Pax6, (K,P) p57, (L,Q) Nrl, (M,R) Pnr, (N,S) Chx10, (O,T) clusterin,

View larger version (35K): [in a new window]   Fig. 8. Clonal inactivation of Notch1 in postnatal RPCs. P0 or P3 Notch1 flox/flox mice retinae were infected in vivo with a replication incompetent retrovirus encoding alkaline phosphatase without or with cre recombinase (pLIA versus pLIA-CRE). (A) Representative clones from a LIA infection including one single rod clone and one bipolar clone. (B,C) Quantitation of cell types resulting from LIA or LIA-CRE infections on Notch1 flox/flox retinae at P0 (B) and P3 (C). Three independent retinae totaling 400-800 clones were analyzed for each timepoint and type of virus. Change in cell types in the LIA-CRE-infected compared with control (LIA-infected retinae) is statistically significant (*P<0.05, **P<0.01, two-tailed t-test assuming equal variances).

View larger version (16K): [in a new window]   Fig. 9. Model of Notch activity in retinal development. Notch is expressed in cycling retinal progenitor cells, where it can be proteolytically processed to form an intracellular fragment, NICD, following its interaction with a Notch ligand. The level of NICD-mediated signal that is finally transduced is read by a progenitor cell to determine the mitotic fate of the daughter cells. If the signal is below a threshold for cycling, at least one postmitotic daughter will be made. If the signal is above physiological levels for cycling, early progenitor cells do not continue to cycle, but they also do not make neurons (Dorsky et al., 1995) (A.P.J. and C.L.C., unpublished). If a postmitotic daughter is made, the level of Notch signaling is a determinate of the fate of the postmitotic daughter. If the Notch signal level is very low, as is the case in a complete loss of function, a photoreceptor is made, probably through activation of Otx2 (Nishida et al., 2003). If the level is intermediate, a non-photoreceptor neuron is made. The type of non-photoreceptor neuron is determined by the combination of homeobox and bHLH proteins present in the progenitor and/or newly postmitotic cell (Hatakeyama and Kageyama, 2004).