Development 142: doi:10.1242/dev.121533: Supplementary Material
Figure S1. Monitoring Dct-Cre activity at postnatal stages. (A-B) EGFP expression in Dct-Cre;Z/EG eyes (P9) detected in both peripheral (A, A') and central regions (B, B') of RPE. In the photoreceptor layer, few cells located in the peripheral optic cup expressed the GFP (7%, SD +-1%, N=4), while in the central retina recombinant cells were not detected. (C) QPCR monitoring the expression of Dicer1 in RPE (P5, N=7, P<0.02) and the neuroretina (P9, N=5, P=n.s.) from Dicer1loxP/loxP;Dct-Cre. Error bars indicate SE. Scale bars: 100µm in A, B; 50µm in A', B'.
Development | Supplementary Material
Development 142: doi:10.1242/dev.121533: Supplementary Material
Figure S2. miRNAs deficient RPE does not effect on ciliogenesis of the photoreceptors cells. (A-B) Scanning electron microscopy images showing the ultrastructure of the photoreceptors from P5 Dicer1loxP/loxP (A) and Dicer1loxP/loxP;DctCre (B) mice. The connecting cilias (cc) are emerging from the inner segments both in the control and mutant RPE. Scale bars: 5µm (A,B); 2µm (A',B').
Development | Supplementary Material
Development 142: doi:10.1242/dev.121533: Supplementary Material
Figure S3. Rhodopsin accumulation and cell death in Dicer1loxP/loxP;Dct-Cre and Dgcr8loxP/loxP;Dct-Cre photoreceptors. (A,C) Rhodopsin (red) is highly present in photoreceptor outer segments in control RPE. (B,D) In the Dicer1loxP/loxP;Dct-Cre RPE and Dgcr8loxP/loxP;Dct-Cre RPE, most of the rhodopsin has been mis-targeted to the outer nuclear layer. (F,H) Cleaved Caspas3 (Ccasp3) is evident in the Dicer1loxP/loxP;Dct-Cre RPE and Dgcr8loxP/loxP;Dct-Cre outer nuclear layer (ONL) as opposed to the control (E,G). Retinal histology (H&E staining) in mutant and control RPE showing acute loss of ONL at P19 (I,J). Abbreviations: ISs, inner segments; OSs, outer segments; RPE, retinal pigmented epithelium. Scale bars: 100 µm.
Development | Supplementary Material
Development 142: doi:10.1242/dev.121533: Supplementary Material
Figure S4. Validation of altered gene expression in the Dicer1 deficient RPE. (A,B) Meis2 and (C,D) Pax6 detected by antibody labeling at P5 control (A,C) and Dicer1 deficient RPE (B,D). (E) QPCR for monitoring expression of miR-204 targets Slc6a8 and Ap1S3 (N=3, P<0.05). Error bars indicate SE. Abbreviations: GCL, ganglion cell layer; NBL, neuroblastic layer; RPE, retinal pigmented epithelium. Scale bar 100 µm.
Development | Supplementary Material
Development 142: doi:10.1242/dev.121533: Supplementary Material
Table S1. Significantly altered miRNA with a cut off: FDR<0.1, P<0.05, FC>1.5
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Table S2. Significantly altered mRNA with a cut off: FDR<0.1, P<0.05, FC>1.5
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Table S3. Putative miRNA-mRNA targets
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Table S4. List of primers used for quantitative real time PCR
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Development | Supplementary Material
Development 142: doi:10.1242/dev.121533: Supplementary Material
Table S5. List of antibodies
Antibody Rabbit anti-Sox9 Rabbit anti-cleaved Caspase3 Sheep anti-Chx10 Mouse antiRhodopsin Rabbit anti-p-Ezrin Mouse anti-Rpe5 Goat anti-GFP Rabbit anti-Cralbp Donkey anti-rabbit conjugated to alexa594 Alexa488 donkey anti mouse Alexa488 donkey anti sheep Alexa488 donkey anti goat
Working concentration 1:200 1:200
Company
Serial number
Chemicon Cell signaling
ab5535 9661
1:1000 1:1000
Exalpha M. Applebury
X1180P
1:250 1:250 1:500 1:1000 1:1000
Cell signaling Lifespan Rockland
3141 LS-B90 600-101-215
Invitrogen
A21207
1:1000
Invitrogen
A21202
1:1000
Invitrogen
A11015
1:1000
Invitrogen
A-11055
Development | Supplementary Material
