Supporting information
Figure legends to Supplementary Figures
Figure S1. The deletion of Notch2 receptor in the RPE results in a small eye phenotype, with no histological effect on the eye structure.
a-c. The disruption of Notch signaling through Tyrp1::Cre-mediated deletion of the Notch2 receptor resulted in a reduced size of the enucleated eyes in Tyrp1::Cre/°; Notch1+/+; Notch2flox/flox (b) and Tyrp1::Cre/°; Notch1flox/flox; Notch2flox/flox (c) animals, in comparison to control (a). However, this did not affect the structure and pigmentation of the eye. d-i. HE-stained eye sections revealed that the structure of the RPE and the neural retina, as well as the presence of the lens and the choroid were not altered by the disruption of the Notch pathway (e,f,h,i), and were similar to control eyes (d,g). Note that Figure S1a and d are equally depicted in Figure 1 (Figure 1g and j). The age of mice is indicated in weeks (w). N1∆ = Tyrp1::Cre/°; Notch1flox, N2∆ = Tyrp1::Cre/°; Notch2flox, ch = choroid, scale bars = 500 µm (d-f) and 50 µm (g-i).
Figure S2. The structure of the RPE and the expression of pigment cell-specific genes are unaffected in Tyrp1::Cre/°; RBP-Jflox/flox eyes.
a,b. HE staining of eye sections at E13.5 showed that the RPE layer deficient in the transcription factor RBP-J was normally developed and pigmented in Tyrp1::Cre/°; RBP-Jflox/flox embryos (b). c,d. Staining of nuclei with Dapi revealed that RPE cells in mutant eyes (d) were arranged in a cell monolayer as in control eyes (c). e,f. In comparison to control embryonic eye sections (e), the deletion of RBP-J in RPE cells did not affect the expression of pigment cell-specific genes, and Mitf (arrowheads), Dct, Tyrp1 and Tyrosinase (Tyr) were expressed normally in Tyrp1::Cre/°; RBP-Jflox/flox eye sections (f). g,h. At 3 weeks, the deletion of RBP-J did not affect the histological appearance of the eye, and HE-stained Tyrp1::Cre/°; RBP-Jflox/flox eye sections showed normal RPE, choroid, and neural retina. i,j. By staining the nuclei with Dapi, we observed that the arrangement of RPE cells in a monolayer remained unaffected in mutant eyes. Although mutant eyes were smaller (see Figure 1), the density of RPE cells (arrowheads) was similar in both control (i) and Tyrp1::Cre/°; RBP-Jflox/flox (j) eyes. The dotted line in immunofluorescence pictures marks the external borderline of RPE cell nuclei. The age of mice is indicated in embryonic days (E) and weeks (w). RBP∆ = Tyrp1::Cre/°; RBP-Jflox, ch = choroid, scale bars = 25 µm.
Figure S3. The Mart1::Cre-mediated deletion of RBP-J in RPE cells results in a small eye phenotype.
a. The RPE-specific expression of the Mart1::Cre transgene was detected in Mart1::Cre/°; Rosa26R/- mice by X-Gal and NFR staining. At E13.5, the Cre-induced recombination of floxed alleles was restricted to RPE cells, and no labeled cells were detected in the neural retina and the ciliary margin. b,c. Disruption of Notch signaling in Mart1::Cre/°; RBP-Jflox/flox RPE cells results in a reduced size of the eyeballs and of the pupil opening (b, control). d-g. On sections, the morphology of the mutant eye structures appeared mostly unaffected and similar to control (d), except for the vitreous, which is absent in the Mart1::Cre/°; RBP-Jflox/flox eye (e). Enlargement of the retinal region (f,g) confirmed that neither the neural retina nor the RPE layer were affected by the disruption of Notch signaling in RPE cells at 4 weeks. The age of mice is indicated in embryonic days (E) and weeks (w). Mart1::Cre = Mart1::Cre/°, R26R = Rosa26R/-, RBP∆ = Mart1::Cre/°; RBP-Jflox, scale bars = 500 µm (d,e) and 100 µm (a,f,g).
Figure S4. The expression of Tyrp1::NotchIC induces an increase in the proportion of BrdU-positive and p27Kip1-negative cells, that is rescued by the deletion of RBP-J.
a. At E13.5, the proportion of proliferating BrdU-positive RPE cells is not affected by expression of the Tyrp1::NotchIC transgene (n = 20 eye sections), nor the deletion of RBP-J in Tyrp1::Cre/°; RBP-Jflox/flox and Tyrp1::NotchIC/°;Tyrp1::Cre/°; RBP-Jflox/flox mice (n = 20 and 15 eye sections). About 20% of RPE cells were proliferating in control (n = 26 eye sections) and transgenic eyes. b. At E15.5, an increase in the number of proliferating RPE cells was observed in Tyrp1::NotchIC/° eyes (23% of BrdU-positive cells, n = 18 eye sections), in comparison to controls (12%, n = 11 eye sections). In addition, deletion of RBP-J rescued the proliferation phenotype induced by Tyrp1::NotchIC expression and lowered the proportion of BrdU-positive RPE cells to 7% (n = 26 eye sections). c. The negative cell cycle-regulator p27Kip1 was expressed in 85% of control RPE cells at 4 weeks (n = 16 eye sections). However, in proliferating RPE of Tyrp1::NotchIC/° eyes, p27Kip1 was only detected in 8% of the RPE cells forming the tumor masses (n = 20 eye sections). The age of mice is indicated in embryonic days (E) and weeks (w). RBP∆ = Tyrp1::Cre/°; RBP-Jflox, NIC = Tyrp1::NotchIC/°, *** p < 0.001.
Figure S5. Tumor cells in Tyrp1::NotchIC/° RPE express Tyrp1 and Dct.
a,b. Tyrp1 immunostaining (red, PEP1) of control RPE cells (a, non-transgenic) and of tumor cells (b, in Tyrp1::NotchIC/° eyes) confirmed the pigment cell origin of the tumor. c,d. Dct immunostaining (red, PEP8) showed similar expression pattern in control (c) and Tyrp1::NotchIC/° (d) eye sections. The dotted line marks the external borderline of RPE cell nuclei. The age of mice is indicated in weeks (w). NIC = Tyrp1::NotchIC/°, scale bars = 50 µm.
Figure S6. RBP-J-dependent Notch signaling activity in RPE tumors affects proliferation and differentiation.
a. In control mice, RPE cells at 4 weeks express RPE65 and Pmel17, but are negative for Pax6. b. The expression of the Tyrp1::NotchIC transgene indicated a more undifferentiated state of RPE cells when cells in tumor mass were negative for RPE65 and positive for Pax6. While unpigmented tumor cells did not express Pmel17, some RPE cells in the pigmented area of the tumor were positive for this marker of premelanosomes. c. Since Notch signals through RBP-J in RPE cells, deletion of the downstream transcription factor RBP-J in Tyrp1::NotchIC/°; Tyrp1::Cre/°; RBP-Jflox/flox eyes rescued the postmitotic state of RPE and cells were positive for RPE65 and Pmel17, while negative for Pax6. The dotted line marks the external borderline of RPE cell nuclei. The age of mice is indicated in weeks (w). RBP∆ = Tyrp1::Cre/°; RBP-Jflox, NIC = Tyrp1::NotchIC/°, scale bars = 50 µm.
Materials and Methods
Genotyping
Genotyping of mice was performed on DNA isolated from tail or ear biopsies using standard PCR buffer composition and reaction (Porret et al., 2006). The Tyrp1::Cre transgene (0.4 kb fragment) was detected by PCR (15 sec at 95°C, 20 sec at 60°C, 35 sec at 72°C, 30 cycles) using primers [Cre26] 5’-CCT GGA AAA TGC TTC TGT CCG-3’ and [Cre36] 5’-CAG GGT GTT ATA AGC AAT CCC-3’. The same conditions were used to identify the Dct::LacZ transgene (0.45 kb) using LacZ-specific primers [LacZ_fwd] 5’-TCG TCT GCT CAT CCA TGA CC-3’ and [LacZ_rev] 5’-GAT TTC CAT GTT GCC ACT CG-3’. The floxed and wild-type alleles of Notch1, Notch2 and RBP-J were detected by PCR (1 min at 93°C, 1 min at 56°C, 1 min at 72°C, 40 cycles). To amplify the floxed (0.37 kb) and the wild-type (0.3 kb) alleles of Notch1, we used the primers [Notch1_fwd] 5’-CTG ACT TAG TAG GGG GAA AAC-3’ and [Notch1_rev] 5’-AGT GGT CCA GGG TGT GAG TGT-3’. Primers [Notch2_fwd] 5’-GAG AAG CAG AGA TGA GCA GAT G-3’ and [Notch2_rev] 5’-CTG AGA TGT GAC ACT TCT GAG C-3’ amplified the floxed (0.3 kb) and wild-type (0.25 kb) alleles of Notch2, while the floxed (0.88 kb) and wild-type (0.72 kb) alleles of RBP-J were detected using the following primers: [RBP-J_fwd] 5’-CTG ACT TAG TAG GGG GAA AAC-3’ and [RBP-J_rev] 5’-AGT GGT CCA GGG TGT GAG TGT-3’. To identify Rosa26R mice, the wild-type (0.6 kb) and mutant (0.3 kb) fragments were identified by PCR using [Rosa26_1] 5’-AAA GTC GCT CTG AGT TGT TAT-3’, [Rosa26_2] 5’-GCG AAG AGT TTG TCC TCA ACC-3’, and [Rosa26_3] 5’-GGA GCG GGA GAA ATG GAT ATG-3’.
Transgenic mice
The Tyrp1::NotchIC construct was generated by cloning first the 1.4 kb Tyrp1 promoter (Jackson et al., 1991; Raymond and Jackson, 1995) from the Tyrp1::LacZ plasmid (Murisier et al., 2006) in a modified pUC18 vector cut with XbaI and XhoI. Separately, a 2 kb BamHI-EcoRI fragment encoding the Notch1 intracellular domain, abbreviated NotchIC and previously inserted in the pMV11 vector digested by SmaI, was ligated to SV40 splice and polyadenylation sequences (EcoRI-EcoRI) into pBluescript II KS (+/-) (Stratagene). The SpeI-KpnI fragment was then inserted downstream of the Tyrp1 promoter, yielding the final Tyrp1::NotchIC plasmid. The Tyrp1::NotchIC fragment was isolated and separated from the vector prior to injection into fertilized oocytes derived from B6D2F1 male and female matings (Porret et al., 2006). Injected oocytes were transferred into pseudopregnant females. Stable lines were obtained by mating transgenic offspring to wild-type C57BL/6 mice. Tyrp1::NotchIC transgenic mice and embryos were identified by PCR (15 sec at 95°C, 20 sec at 60°C, 35 sec at 72°C, 30 cycles). A 0.46 kb fragment was generated by amplification of genomic DNA from tail or ear biopsies using the following primers: [Tyrp1_left 2] 5’-CCA TCA CAA GGA AAC CAG TG-3’ and [NotchIC_right 2] 5’-TTG GTC TCC AGG TCT TCG TC-3’.
RPE isolation for DNA and RNA extraction
Eyeballs were gently enucleated after cutting the optic stalk, and washed in PBS. Eyes were then treated for 30 min in Dispase II (2 mg/ml, Sigma) at 37°C. Using injection needles (BD Microlance 3 Gauge 18 and 21), the sclera was detached without disruption of the eyeball. Then, making an incision, the lens and the anterior part containing the iris were removed. The RPE was then isolated from the neural retina pulling both tissues in opposite directions. DNA was then extracted from isolated RPE, and analyzed by PCR. The deleted (delta) Notch2 fragment (0.4 kb)resulted from amplification with primers [Notch2del_fwd] 5’-CAG TTC ACA GGG AGA AGC AG-3’ and [Notch2del_rev] 5’-GTG CAC ATA TGC CTT AGC-3’, in the same conditions as for floxed and wild-type Notch2 alleles. For RT-PCR analysis, total RNA was isolated from frozen RPE samples using Trizol reagent (Invitrogen). cDNA from total mRNA was synthesized using oligo-dT primers and SuperScript® III Reverse Transcriptase (Invitrogen). The expression of the Tyrp1::NotchIC transgene was analyzed by detecting the amplified cDNA fragment in the SV40 splice and polyadenylation (pA) sequences, using primers [pA_fwd] 5’-CCT TAC TTC TGT GGT GTG AC-3’ and [pA_rev] 5’-GAG GAG TAG AAT GTT GAG AGT-3’. The amplified cDNA (0.2 kb) is distinguishable from genomic DNA (0.26 kb), due to the presence of a 60 bp-intron.
X-Gal staining of eye sections
Enucleated eyeballs were embedded in OCT, frozen on dry ice and kept at -70°C. Cryosections (8 µm) were fixed (3 min, 4% paraformaldehyde in PBS), washed three times with PBS, and treated twice in permeabilization solution (2min, 0.1 M phosphate buffer pH 7.3, 2 mM MgCl2, 0.01% desoxycholate, 0.02% NP40). Sections were then stained (3.33 mM potassium ferricyanid, 3.33 mM ferrocyanid, 20 mM Tris HCl pH 7.4, 0.66 mg/ml X-Gal in permeabilization solution) overnight at RT (protected from light). Sections were finally counterstained with Nuclear Fast Red (NFR), and mounted in Eukitt.
Immunohistochemistry
Isolated embryos or eyes were fixed in 4% paraformaldehyde for 1 hour on ice before embedding in paraffin and sectioning (4 µm). After rehydratation of the sections through xylol and ethanol, antigen retrieval was performed in 10 mM Tris-EDTA (pH 9) for 20 min at 95°C. After washing in phosphate buffered saline (PBS), the sections were incubated for 30 min in a blocking solution (BSA 2%, Tween 0.05%, goat serum 1%, in PBS) at RT, and then overnight with diluted primary antibody at 4°C. Tissue sections were then treated with the secondary antibody conjugated to a fluorescent dye (45 min, 37°C), and counterstained on slides with DAPI (4’-6-Diamidino-2-phenylindole, 1:4000) for 5 min at RT. Slides were mounted in DABCO-glycerol (Sigma D-2522). Paraffin sections and antibody stainings were performed according to standard procedures. Primary antibodies and dilutions were as follows: rabbit anti-Dct (provided by V. Hearing, PEP8; 1:1000), rabbit anti-Mitf (provided by S. Saule, 1:1000), rat anti-mouse BrdU (1:200), mouse anti-human Ki67 (1:500), rabbit anti-NotchIC (ABCAM, #ab8925; 1:100), mouse anti-p27Kip1 (BD Transduction Laboratories, #610241; 1:100), mouse anti-human RPE65 (Santa Cruz, sc-53489; 1:1000), mouse anti-chicken Pax6 (Developmental Studies Hybridoma Bank; 1:500), rabbit anti-human Pmel17 (provided by M. Marks, Pep13h; 1:1000 (Bersonet al., 2001)), rabbit anti-mouse Pmel17 (provided by M. Marks,mPmel-N; 1:1000 (Theoset al., 2006)). Alexa Fluor® conjugated secondary antibodies (Molecular Probes) and dilutions were as follows: goat anti-rat IgG Alexa 488 (1:1000), donkey anti-rabbit IgG Alexa 568 (1:1000), goat anti-mouse IgG Alexa 488 (1:1000).
BrdU labeling at embryonic stages
Pregnant mice were injected intraperitoneally with BrdU (5-bromo-2-deoxyuridine, 10 mg/kg body weight, Sigma) 24 hours and 2 hours before sacrifice. Embryos were handled and sectioned as described for immunohistochemistry analysis. Using a rat anti-mouse BrdU (1:200) primary antibody, eye sections were labeled following the protocol used for immunofluorescence experiments.
References
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