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DVDY_23762_sm_SuppFig1.tif425KSupporting Information Figure 1. A: Quantification of the proportion of AP-2-positive cells that express Ki67 on E16.5 retinal sections, based on scoring of 2,245 AP-2α-positive cells, 2326 AP-2β-positive cells and 1,143 AP-2γ-positive cells. Bars represent the percentages for a particular AP-2 protein that co-stains with Ki67: 1.47 ± 0.24 (AP-2α), 1.88 ± 0.21 (AP-2β) and 1.51 ± 0.36 (AP-2γ). B: Quantification of the proportions of PAX6-positive INL cells that express AP-2γ compared with AP-2α, at the stages indicated. The GCL was excluded from that analysis, given that PAX6-positive ganglion cells are present. At P0, brightly labeled PAX6-positive cells in both the INL and onbl (presumptive amacrine cells) were counted. At P7 and P28, all PAX6-positive INL cells except horizontal cells were included. At all stages examined, the proportion of PAX6-positive INL cells expressing AP-2γ was significantly smaller than the proportion expressing AP-2α; however the relative proportions of PAX6-positive cells expressing AP-2γ versus AP-2α significantly differed between stages. Bars represent the mean ±SEM of counts from 3 animals. ***P < 0.001; **P < 0.01.
DVDY_23762_sm_SuppFig2.tif1518KSupporting Information Figure 2. A: Schematic diagram of crosses used to generate Tcfap2a/b mutants. Primer annealing sites for wild-type and mutant alleles are depicted as colored arrows. Note that, in the final cross of the breeding scheme, one mouse in the breeder pair is always homozygous for the AP-2α floxed allele (Tcfap2alox/lox) so that all offspring will inherit one Tcfap2alox allele – therefore the only Tcfap2a polymerase chain reaction (PCR) required on offspring from the final cross determines whether the Tcfap2aki7lacZ versus wild-type (Tcfap2a+) allele is present. See text of Materials and Methods section for remaining details. B: Immunostaining of wild-type (top) and Tcfap2a/b mutant (bottom) retinas at embryonic day (E) 18.5. Labeling with anti-AP-2α (3B5; green) shows loss of AP-2α from the peripheral Tcfap2a/b mutant retina, as expected upon α-Cre-mediated excision. Labeling with anti-AP-2β (Cell Signaling #2509; red) shows loss of AP-2β from the entire Tcfap2a/b mutant retina, as expected based on the homozygous germ-line KO allele. Note that antibodies do not cross-react with other AP-2 family members. Dotted line denotes retina. P, peripheral retina. Scale bars = 100 μm.
DVDY_23762_sm_SuppFig3.tif5965KSupporting Information Figure 3. Horizontal cells are present in central retina of Tcfap2a/b mutants where α-Cre activity diminishes, but absent from the periphery. A: Horizontal section of P0 Tcfap2a/b mutant retina co-immunostained with anti-AP-2α (green) and anti-PGP9.5 (red), counterstained with DAPI (4,6-diamino-2-phenylindole; blue). As a result of the breeding scheme used to generate Tcfap2a/b mutants, the genotypes of the central and peripheral retina are equal with respect to lack of Tcfap2b function (i.e., two null alleles), and only differ by one allele of Tcfap2a (i.e., intact versus recombined floxed allele). Note the remaining AP-2α protein in the central mutant retina, as well as the presence of AP-2α/PGP9.5 double-labeled horizontal cells in this area (arrowheads). B: Horizontal sections of X-gal stained E18.5 control (left) and Tcfap2a/b mutant (right). Arrowheads point to X-gal-positive cells in the presumptive horizontal cell layer of the control retina, which are absent from the double mutant. Scale bars = 100 μm.
DVDY_23762_sm_SuppFig4.tif2419KSupporting Information Figure 4. No changes in proliferative markers or apoptosis were detected in Tcfap2a/b mutant retinas. A–F: Horizontal sections of embryonic day (E) 18.5 control (A,C,E) and Tcfap2a/b mutant (B,D,F) peripheral retinas immunolabeled with anti-PH3 (A,B; red), anti-PCNA (C,D; green), and anti-Cyclin D1 (E,F; red), and counterstained with DAPI (4,6-diamino-2-phenylindole; blue). G,H: Apoptotic cells (green) detected using the TUNEL (Terminal uridine deoxynucleotidyl transferase-mediated dUTP Nick End Labeling) assay on horizontal sections of E18.5 control (G) and Tcfap2a/b mutant (H) peripheral retinas counterstained with DAPI (blue). Scale bars = 100 μm.
DVDY_23762_sm_SuppFig5.tif2103KSupporting Information Figure 5. Expression patterns of additional retinal markers in Tcfap2a/b mutants resemble those of control littermates. A–H: Horizontal sections of P0 control (A,C,E,G) and Tcfap2a/b mutant (B,D,F,H) peripheral retinas immunolabeled (red) with anti-POUF1/2/3 (A,B), anti-OTX2 (C,D), anti-CRX (E,F), or anti-VSX2 (G,H), and counterstained with DAPI (4,6-diamino-2-phenylindole; blue). Scale bars = 100 μm.

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