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Supplemental Figure 1. Recombination Pattern of Six3-Cre in the Adult Retina and Brain

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Supplemental Figure 1. Recombination Pattern of Six3-Cre in the Adult Retina and Brain Supplemental Figure 1. Recombination pattern of Six3-cre in the adult retina and brain. The conditional reporter line, R26R, commences expression of β-galactosidase upon Cre-mediated recombination. The recombination pattern of the Six3-cre transgene in the retina on P21 reveals widespread recombination in all layers (A-C). At the retinal margin, areas devoid of recombination are apparent, as depicted by gaps in the X-gal staining pattern in this region (C, arrowheads). Co- immunolabeling using anti-Isl1 and anti-β-galactosidase reveals that most Isll+ cells in the INL co-localize with Six3-cre's lineage (D-F), although several Isl1+ cells are devoid of detectable reporter expression (D-F, arrowheads). At the retinal margin, many more Isl1+ cells devoid of detectable reporter expression are encountered (G-I, arrowheads). The recombination pattern of Six3-cre in the brain of P21 animals reveals widespread recombination in the hypothalamus, septum and striatum (J). Scale bar equals 50 µm in B (applies to B-C), in F (applies to D-F), in I (applies to G-I), and 1mm in J. Supplemental Figure 2. Additional cell marker expression in the Isl1-null retina. Isl1-null retinas display a 71% reduction in the expression of the RGC marker Pou4f1 (B) compared to control (A; average number of Pou4f1+ cells ± standard deviation: controls: 35 ± 7, n=3; Isl1-nulls: 10 ± 2, n=3, p<0.01, Student's t test). A slight 17% increase in the numbers of the horizontal cell marker, Calbindin-28K, in Isl1-null retinas is observed, although this change is not significant (compare C to D; average number of Calbindin-28K+ cells ± standard deviation: controls: 10 ± 2, n=4; Isl1-nulls: 12 ± 1, n=4, p=0.05, Student's t test). A 27% reduction in the Müller cell marker, Cdkn1b, is observed in the Isl1-null retina (F) compared to control (E; average number of Cdkn1b+ cells ± standard deviation: controls: 41 ± 4, n=3; Isl1-nulls: 30 ± 4, n=3, p<0.05, Student's t test). Toluidine blue stained sections revealed equivalent numbers of rods and cones in Isl1-null retinas (H) compared to controls (G), of which representative examples are highlighted by black and white arrowheads, respectively (average number of rods ± standard deviation: controls: 663 ± 28, n=3; Isl1-nulls: 660 ± 49, n=3, n.s., Student's t test; average number of cones ± standard deviation: controls: 30 ± 5, n=3; Isl1-nulls: 31 ± 2, n=3, n.s., Student's t test). Scale bar equals 50 µm in F (applies to A-F) and in H (applies to G-H). Supplemental Figure 3. Remaining reporter-positive cells in Isl1-null retinas co-localize with residual Isl1 expression. Immunolabeling using anti-β-galactosidase demonstrates a substantial loss of reporter-positive cells in Isl1-null retinas (D) compared to controls (A). The remaining Isl1- immunoreactivity co-localizes with the remaining reporter gene expression (D, overlay in F), and represents incomplete deletion of Isl1 using the Six3-cre transgene in Isl1-nulls. Scale bar equals 50 µm in F (applies to A-F). Supplemental Figure 4. Partial retinal ganglion cell loss alone does not affect bipolar subtype development. Confocal micrographs of rod and cone bipolar marker expression in mature retinas from Pou4f2-null and control littermates (n=3). Prkca+ rod bipolar have similar characteristics in Pou4f2-null and controls, with prominent, high-intensity staining of rod bipolar endfeet in both (arrowheads in A and B). The number of rod bipolars is not significantly different in Pou4f2-null retinas compared to controls (average number of Prkca+ cells ± standard deviation: controls: 63 ± 6, n=3; Pou4f2-nulls: 55 ± 3, n=3, n.s., Student's t test). Isl1+ bipolar cells, which identify rod bipolar and ON-cone bipolar cells, are also not significantly different in Pou4f2-nulls compared to controls (upper brackets in C and D; average number of Isl1+ bipolar cells ± standard deviation: controls: 108 ± 6, n=3; Pou4f2-nulls: 113 ± 10, n=3, n.s., Student's t test). Isl1+ cholinergic amacrine cells are not significantly different either (lower brackets in C and D; average number of Isl1+ amacrine cells ± standard deviation: controls: 17 ± 6, n=3; Pou4f2-nulls: 15 ± 6, n=3, n.s., Student's t test). CABP5+ bipolar cells are not significantly changed in Pou4f2-nulls compared to controls (E and F; average number of CABP5+ cells ± standard deviation: controls: 111 ± 10, n=3; Pou4f2-nulls: 105 ± 22, n=3, n.s., Student's t test). CABP5+ endfeet from OFF cone bipolar, ON cone bipolar and rod bipolar cells are all detectable in Pou4f2-null retinas (arrowheads in F). The OFF cone bipolar marker, Vsx1, is not significantly changed in Pou4f2-null retinas (H) compared to control (G; average number of Vsx1+ cells ± standard deviation: controls: 40 ± 1, n=3; Pou4f2-nulls: 45 ± 14, n=3, n.s., Student's t test). Anti-Pou4f2 immunolabeling in Pou4f2-nulls confirms 'null' status of animals as no detectable Pou42 labeling is observed (J). Scale bar equals 50 µm in J (applies to A-J). Supplemental Figure 5. Co-localization of remaining Isl1+ cells with markers of bipolar and retinal ganglion cells in the Isl1-null retina. Co- immunolabeling using anti-Isl1 with various cell type markers were performed to assess the fate of remaining Isl1-expressing cells in the Isl1-null retina. A predominant fraction of remaining Isl1+ cells (B) co- localizes with the pan-bipolar marker Chx10 (C, arrowheads in C). Isl1+/Chx10- negative cells (C), positioned lower in the INL represent amacrine cells. As in the mature wildtype retina, Isl1+ bipolar cells co- localize with ON-bipolar markers, such as Goα (D-F, arrowheads in F). The ON/OFF-bipolar marker CABP5 partially co-localizes with Isl1+ cells p(G-I). CABP5+ OFF-bipolar cells terminating nearest to the INL (asterisk in G and I) do not co-localize with Isl1, and CABP5+ ON-bipolar cells terminating closer to the GCL (white arrowheads in G-I) did co-localize with Isl1. Some Isl1+/CABP5- cells are also observed (yellow arrowheads in G-I). The ganglion cell marker Pou4f1 co-localizes with residual Isl1+ cells in the GCL (J-L). Scale bar equals 50 µm in L (applies to A-L). Supplemental Figure 6. Immunolabeling for cleaved Caspase-3 in Isl1- null retinas. Fluorescence micrographs of anti-activated caspase-3 immunolabeling are shown for control and Isl1-null retinas from P5-P7. Caspase-3+ cells in the INL were scored at these different ages. While numbers of caspase-3+ cells at P5 were not significantly different (compare B to A; n=3), there was a significant reduction in the number of caspase-3+ cells at P6(n=3) and P7(n=3) in Isl1-nulls compared to controls (compare D to C and F to E). Summary of quantitation is shown in G. Scale bar equals 50 µm in F (applies to A-F). *p<0.05 Supplemental Figure 7. Residual Isl1 expression fails to establish Prkca+ rod bipolar with 'wildtype' morphology. 10 µm stacked confocal images of Prkca-labeled rod bipolar cells in control, Pou4f2-null and Isl1-null retinas were traced in Photoshop and analyzed subsequently in Image J. Anti-Prkca and anti-Isl1 co-immunolabeling was performed in Isl1-null retinas to document Isl1 expression status. Areas of dendritic and terminal arborizations of Prkca+ rod bipolars in Isl1-null retinas are significantly decreased compared to control cells (compare C to A, summarized in D and E), except at the peripheral margin, where a clustering of Isl1+ cells is observed (compare C to A, summarized in D and E). Pou4f2-null rod bipolar cells on the other hand do not significantly differ from control cells in this respect (compare B to A, summarized in D and E). The length of the bipolar cell on the other hand is significantly decreased in both Isl1-null and Pou4f2-null retinas compared to control (compare B and C to A, summarized in F). Two- tailed Student t tests were performed adjusting the significance level to 0.017 from 0.05 because of the multiple comparisons. ****p<0.0001, ** p<0.01, n.s. p0.017. Control (n=6 cells); Pou4f2-null (n=5 cells); Isl1- null (n=14 cells from central retina; n=5 cells from peripheral retina). Supplemental Figure 8. A reduction in late retinal progenitor proliferation does not accompany the reduction in restricted late born cell types in the Isl1-null retina. Fluorescence micrographs of anti-BrdU immunolabeling following 30-minute BrdU pulses at P5 and 7 in controls (A,C,E,G) and Isl1-null retinas (B,D,F,H) were performed to gather whether reductions in proliferation of late retinal progenitors in mutants could underlie later reductions in cell numbers. While a large number of BrdU- incorporating cells are encountered in the peripheral retina of control (A) and Isl1-null retinas at P5 (B), markedly fewer BrdU+ cells are encountered in both controls (C) and Isl1-nulls centrally (D). The number of BrdU+ cells in Isl1-nulls, however, is greater in the central retina at P5 (number of BrdU+ cells: controls: 29 and 18, n=2; Isl1-nulls: 64 and 62, n=2). By P7, a marked down regulation of BrdU+ cells peripherally is observed as well, as evidenced by a trailing edge of BrdU+ cells at the peripheral margin (arrowheads in E and F). Despite the general reduction in BrdU+ cells in controls and Isl1-nulls, the trailing edge of BrdU+ cells in the periphery extends more centrally in mutants at P7, suggesting the general reduction in proliferation is delayed in the Isl1-null retina.
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