DEV010751 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

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• Supplemental Figure S1 -

  Fig. S1. Expression of Isl1 and Isl2 during retina development. (A-D) In situ hybridization of wild-type retina sections with Isl1 and Isl2 probes. Compared with Isl1 expression in RGCs and amacrine cells (arrows) (A,C), Isl2 is expressed only in selective 30% RGCs in the GCL (B,D). (E-J) Immunolabeled retinal sections at E13.5 (E-G) and E15.5 (H-J) show the overall co-expression (arrows) of ISL1 (red) and BRB3B (green) in Isl2-null mice with occasional BRN3B+-only cells (open arrowhead). The anti-ISL1 immunolabeling shows an identical ISL1 expression pattern in wild-type (see Fig. 1) and Isl2-null retina. Insets show the enlarged views of the corresponding boxed regions. NBL, neuroblast layer; GCL, ganglion cell layer. Scale bar: 100 µm.

• Supplemental Figure S2 -

  Fig. S2. Isl1-lacZ reporter expression recapitulates the expression pattern of endogenous Isl1. In situ hybridization, anti-ISL1 immunolabeling and X-Gal staining of Isl1^lacZ/+ retina sections at E14.5 show the similar expression profile of Isl1 mRNA (A), ISL1 protein (B) and Isl1-lacZ (C). Insets show the enlarged views of the corresponding boxed regions. Scale bar: 100 µm.

• Supplemental Figure S3 -

  Fig. S3. Co-occupancy of ISL1 and BRN3B on RGC promoters in E13.5 wild-type retina but not in Brn3b- null or Isl1-null tissues. (A) Anti-BRN3B and anti-ISL1 antibodies co-precipitate with the promoters of Brn3b, Shh, Brn3a and Isl2 in E13.5 wild-type retina. Both antibodies fail to precipitate with these promoters in Brn3b-null retina. Anti-ISL1 antibody does not precipitate with these promoters in cerebellum. (B) Western blot with anti-ISL1 antibody using cerebellum and retina tissue preparation. The antibody primarily recognizes a single band of ISL1 in retina but not in cerebellum, suggesting that ISL1 is not expressed in cerebellum and that the anti-ISL1-precipitated chromatin is specifically ISL1-bound DNA.

• Supplemental Figure S4 -

  Fig. S4. Progenitor proliferation in E17.5 Isl1-null retina. (A-D) Isl1-null retina displays a slight 17% decrease of M-phase proliferating cells as labeled by anti-pH3 (green, compare A with B; average number of pH3+ cells±s.d.; controls, 184.25±9.19; Isl1-nulls, 152.38± 6.54, n=2, Student’s t-test P<0.05.). (C,D) Enlarged views of the boxed regions in A,B.