Supporting Information
Figure S1  Deleting E2f1, but Not E2f2 or E2f3, Rescues Ectopic Division and Cell Death in P0 and P18 Rb KO Retina
Horizontal sections of the indicated genotypes and ages were stained for nuclei (DAPI, blue), and (A) S-phase (anti-BrdU, red) or (B) apoptosis (TUNEL, red). In Rb −/− retinas, BrdU+ cells extend beyond the normal boundaries at P0 (arrows), and ectopic DNA synthesis continues in multiple layers at later stages. Scale bar is 50 μm. The NBL is where dividing RPCs are located.
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Figure S2  Deleting E2f1 Rescues Ectopic Division and Apoptosis in the Embryonic Rb KO Retina
Horizontal sections of the indicated genotypes and ages (E14 and E16, the period during which SACs are born) were stained for nuclei (DAPI, blue), and either S-phase (upper two panels, anti-BrdU, red) or apoptosis (lower two panels, TUNEL, red). In Rb −/− retinas, BrdU+ and TUNEL+ cells can be seen in the inner retina (arrows). Inactivation of E2f1 rescued these defects. Scale bar is 50 μm. The NBL is where dividing RPCs are located.
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Figure S3  Deleting E2f1, but Not E2f2 or E2f3, Rescues Ectopic Mitosis in the Rb KO Retina
(A) Horizontal retinal sections of the indicated genotypes and ages were stained for nuclei (DAPI, blue) and M-phase (anti-PH3, red). Scale bar is 50 μm.
(B) Quantification of all PH3+ cells.
(C) Quantification of ectopic PH3+ cells.
Error bars represent standard deviation (SD), and asterisks indicate significant difference between retina of WT and indicated genotypes (*, p <0.05; **, p <0.01; ANOVA and Tukey HSD test).
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Figure S4  Deleting E2f2 or E2f3 Does Not Rescue Ganglion, Rod, or Bipolar Cell Death in the Rb KO Retina
(A) Horizontal retinal sections from mice of the indicated ages and genotypes were stained for nuclei (DAPI, blue) and markers that detect ganglion cells (Pou4f2, red), rods and cones (Sag [rod arrestin], green), and rod bipolar cells (Prkca, green). Scale bar is 50 μm.
(B) Quantification of total ganglion (Pou4f2+) cells.
(C) Quantification of total rod bipolar (Prkca+) cells.
(D) Thickness of the ONL, which represents the number of rods.
Error bars represent SD, and asterisks indicate significant difference between retina of WT and indicated genotypes (**, p <0.01; ANOVA and Tukey HSD test).
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Figure S5  E2f1 Deletion Rescues α-Cre;RbloxP/loxP Retinal Function
ERGs were recorded from the indicated genotypes under light adapted (photopic) conditions.
(A) Intensity series.
(B) The b-wave amplitudes as a function of the logarithm of the flash intensity.
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Figure S6  Differentiation Defects in Rb KO SACs
Horizontal retinal sections of indicated genotypes and ages were stained for nuclei (DAPI, blue) and Calb2 ([A], red; only densely stained cells were counted for Figure 3C), Camk2a ([B], green), and Slc18a3 ([C], red). Scale bars are 50 μm.
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Figure S7  GABA Neurotransmitter in the Rb KO Retina and Abnormal SACs in Chx10-Cre;RbloxP/loxP Retina
Horizontal sections of the indicated genotypes and ages of retina were stained for nuclei (DAPI, blue), and (A and B) GABA (red) and Slc18a3 (green) or (C) Chat and Slc18a3 (red).
(A) In P18 WT retina, GABA labelled four IPL tracks, of which the two inner tracks co-stained with Slc18a3. The latter tracks disappeared in the Rb KO retina, and were rescued by E2f3 KO but not E2f1 KO.
(B) At the boundary of the WT (central) and Rb KO area (peripheral retina) the inner GABA+ SAC tracks can be seen disappearing towards the periphery (left).
(C) Slc18a3 staining in the IPL of Chx10-Cre;RbloxP/loxP retina is consistent with the mosaic pattern of Rb inactivation.
Scale bars are 50 μm.
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Figure S8  Subcellular Distribution of E2f3a Isoform in the Developing Retina
Nuclear and cytoplasmic extracts from an equivalent number of retinal cells from mice of the indicated genotypes and ages were analyzed by Western blotting to detect the E2f3a protein. Lysates from E2f3a−/− mice of matched ages were used as a control to confirm the location of E2f3a protein. C, cytoplasmic extracts; N, nuclear extracts.
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Table S1  List of Antibodies and Marker Patterns in Rb/E2f1 DKO SACs
(97 KB DOC)
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Table S2  Real-Time RT-PCR Primers
(49 KB DOC)
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Accession Numbers
The GenBank (http://www.ncbi.nlm.nih.gov/genbank) accession numbers for the major genes and gene products discussed in this paper are Camk2a (NM_009792), Chat (NM_009891), E2f1 (NM_007891), E2f2 (NM_177733), E2f3 (NM_010093), Rb (NM_009029), and Slc18a3 (NM_021712).