DEV02664 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Absence of Bhlhb5 expression in selective retinal cell types. (A-I′′) Retinal sections at P28 were double-immunolabeled with anti-Bhlhb5 (red) and subtype-specific markers (green) as indicated. No Bhlhb5 expression was detected in RGCs (Brn3a⁺, A-A′′), cholinergic amacrine (Isl1⁺ and ChAT⁺, B-B′′ and C-C′′, respectively), dopaminergic amacrine (TH⁺, D-D′′), calretinin⁺ amacrine (E-E′′) and AII amacrine (parvalbumin⁺, F-F′′), rod bipolar (PKCα⁺, G-G′′), horizontal (calbindin⁺, H-H′′) and Müller (cyclin D3⁺, I-I′′) cell types. Scale bar: 50 μm.

• Supplemental Figure 2 -

  Fig. S2. Targeted deletion of Bhlhb5. (A) Bhlhb5 genomic structure, restriction enzyme map and targeting strategy. The DNA fragment containing the Bhlhb5 open reading frame (ORF) region was replaced with the reporter ß-galactosidase (lacZ) gene in the targeting vector. Neo, PGK-neo cassette; TK, MC1-TK cassette. (B) Southern blotting and PCR genotyping of a typical litter from heterozygous to heterozygous mating. The 5′ Southern probe detects 8.1 kb wild-type and 5.1 kb mutant XbaI fragments. PCR genotyping primer sets reveal 623 bp wild-type and 404 bp mutant bands.

• Supplemental Figure 3 -

  Fig. S3. Quantitation of retinal cells immunoreactive for cell type-specific markers. Sections from P21 mouse retinas were immunolabeled with subtype-specific markers and nuclear-counterstained with PI. The number of specific retinal cell types was scored per retinal section area, except that Pax6⁺ and Chx10⁺ cells were counted per 1500 μm length of retinal section. Each histogram represents the mean±s.d. for three or more retinas.

• Supplemental Figure 4 -

  Fig. S4. Effect of Bhlhb5-null mutation on programmed cell death and cell proliferation. (A-D) Retinal sections from Bhlhb5^−/− and wild-type control retinas at indicated developmental stages were stained with anti-activated caspase 3 (green). Compared with the control retina (A,C), no overt change in the number of apoptotic cells (arrowheads) was seen in Bhlhb5-null retina (B,D) at E17.5 and P10. (E-H) Anti-BrdU (E,F, green) and anti-phosphorylated histone H3 (PH3) (G,H, green) labeling experiments show proliferating cells in S- and M-phase, respectively. (I,J) Quantitation of retinal cells immunoreactive for anti-BrdU (I) and anti-PH3 (J). The BrdU+ number was scored per 350 μm length of retinal section and PH3+ number per retinal section. Each histogram represents the mean±s.d. for three retinas. Scale bars: 200 μm.

• Supplemental Figure 5 -

  Fig. S5. Upregulation of Bhlhb5 expression in the absence of Math5. Retinal sections from wild-type (A-F) and Math5-null (G-L) mice at E11.5 to P0 were immunolabeled with anti-Bhlhb5 (green) and nuclear-counterstained with PI (red). (M) RT-PCR analysis shows the increased expression of Bhlhb5 mRNA in the absence of Math5 at E12.5 to P0. β-actin mRNA serves as an internal control. Scale bar:100 μm.