DEV01560 Supplementary Material

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• Supplemental Figure 1 -

  Fig. S1. (A-P) Retinal explant cultures of E18.5 Dlx1/Dlx2 mutant and wild-type retinas cultured for 7 days. All explants feature double retinal layers with the outer retina located at the top and bottom of the sections and the inner retinal layers in the middle of tissue sections (see dividing line A). (A-H) Late-born retinal cell classes were identified on the basis of marker expression. Cone photoreceptors [A,B; peanut agglutinin (PNA)], rod photoreceptors (C,D; Rho4d2), rod bipolar cells (E,F; Chx10) and cone bipolar cells (G,H; Vsx1, arrows) appear unaffected in Dlx1/Dlx2 mutants on the basis of marker expression. Analysis of the early-born cell classes characterized in the E18.5 retinas was also performed. (I-L) Amacrine cells (I,J, syntaxin; K,L, calretinin) and horizontal cells (O,P, Prox1; Q,R Nf165) remain unaffected in retinal explants of Dlx1/Dlx2 mutants. Scale bars: 50 µm in G,H,O,P.

• Supplemental Figure 2 -

  Fig. S2. Characterization of retinal ganglion cells in E18.5 Dlx1/Dlx2 mutant and wild-type retinas by ISL1 expression. (A) Quantification of ISL1-expressing RGCs in Dlx1/Dlx2 mutant retinas compared with paired wild-type littermates showed a significant (t=7.74, P<0.05, n=5) decrease in the number of RGCs in the mutants (A, asterisk). This represented a 39% decrease, on average, in the number of RGCs in the Dlx1/Dlx2 mutant retinas. Qualitatively, ISL1 immunostaining demonstrates a histologically thicker RGL in wild-type retinas (B) than in mutants (C). ISL1+ cells are readily discerned in both mutant and wild-type retinas (B,C, arrows). (D) Negative antibody control. Scale bar: 100 µm in D.

• Supplemental Figure 3 -

  Fig. S3. Quantification of proliferating cells in embryonic Dlx1/Dlx2 mutant and wild-type retinas. Values represent pooled counts from positionally and histologically matched sequential sections from paired mutant and wild-type retinas to completely survey each retina. (A) There were no significant differences identified at E13.5, E16.5 or E18.5 in the number of cells in M-phase as determined by phosphohistone H3 expression (E13.5 t=0.76, P>0.05, n=5; E16.5 t=1.17, P>0.05, n=5; E18.5 t=0.26, P>0.05, n=5). (B) Similarly, no significant differences in the number of cells in S-phase as determined by BrdU pulse labelling could be identified (E13.5 t=0.97, P>0.05, n=5; E16.5 t=0.56, P>0.05, n=5; E18.5 t=0.86, P>0.05, n=5).

• Supplemental Figure 4 -

  Fig. S4. Qualitative characterization of apoptosis by TUNEL assay in Dlx1/Dlx2 mutant and wild-type retinas. TUNEL staining was performed on Dlx1/Dlx2 mutant and paired wild-type littermates retinas at E13.5 (A-C), E16.5 (D-F) and E18.5 (G-I). Results from the TUNEL assay mirrored those generated with antibodies to activated caspase 3 (Fig. 4). At E13.5, apoptotic cells are identified in both wild-type (A, arrows) and mutant retinas (B, arrows). However, more apoptotic cells are evident in mutant retinas (B, arrows). At E16.5, there is more TUNEL staining in mutant retinas (E, arrows) than in wild-type retinas; however, the difference is less pronounced than at E13.5. No differences were apparent at E18.5 (G,H). Scale bars: 200 µm in C; 100 µm in F,I.