(A) Morphology of wild-type and rw337 mutant embryos at 4 dpf. rw337 mutant embryos have smaller eyes compared to wild type at 4 dpf (red arrow). The black arrow shows the cloudy tectum in rw337 mutant embryos, representing cell death. Scale bars: 100 μm. (B) Plastic sections of wild-type and rw337 mutant retinas during development. Higher magnification images of green squares in the left columns are shown in the right columns. Retinal neuroepithelium is normal in rw337 mutants until 2 dpf. However, many round cells reminiscent of mitotic cells are accumulated in the apical region of rw337 mutant retinas at 2 dpf (open green arrowheads), suggesting mitotic defects in retinal progenitor cells. In wild-type retinas, three nuclear layers (RGCL, INL, and ONL) and two plexiform layers (IPL and OPL) are formed at 2.5 dpf. INL consists of amacrine cells (ACs), bipolar cells (BPs), and horizontal cells (HCs). ONL consists of two types of photoreceptors (PRs), rods and cones. However, only RGCs appear near the lens and the outer region was occupied by pyknotic nuclei, indicating dead cells in rw337 mutants. At 3 dpf, INL is formed, but the outer region is still occupied by pyknotic nuclei in rw337 mutants. At 4 dpf, pyknotic nuclei are reduced in rw337 mutants; however, ONL and OPL are absent despite the small number of photoreceptor-like columnar cells in putative INL (PR, asterisks). At 3 and 4 dpf, the CMZ is maintained in rw337 mutants (green double asterisks). (C) The rw337 mutation is mapped in the genomic region flanked by two polymorphic markers, 070702B and zC93F2D, on chromosome 25. This genomic region contains four genes, including banp. (D) banp cDNA consists of 13 exons. In rw337 mutants, T at the donor splice site of the intron between exons 4 and 5 (intron4/5) is converted to G, leading to the insertion of 52 nucleotides (red region) from intron 4/5. A new stop codon appears within this insertion. (E) Wild-type Banp protein has a coiled-coil domain, a BEN domain and a DNA-binding domain. An NLS domain, an RS-rich domain, and MAR-binding domain are shown. A truncated protein missing BEN and DNA-binding domains is predicted in rw337 mutants. (F) Retinal sections of wild-type, wild-type embryos injected with banp-5misMO, and banp MO. banp morphant retinas have similar phenotypes to banprw337 mutants, with increased cell death and loss of the ONL. Yellow open arrowheads show pyknotic nuclei. (G) Retinal sections of wild-type retinas, banprw337 mutant retinas, and banprw337 mutant retinas overexpressing Banp(wt)-EGFP and EGFP-Banp(rw337). Overexpression of Banp(wt)-EGFP inhibits cell death and recovers retinal lamination in banprw337 mutants, whereas overexpression of EGFP-Banp(rw337) does not inhibit retinal defects in banprw337 mutants. Yellow open arrowheads indicate pyknotic cells in banprw337 mutant retinas and banprw337 mutant retinas overexpressing EGFP-Banp(rw337). Scale bars: 20 μm for (B, F, G).RGC, retinal ganglion cell; AC, amacrine cell, BP, bipolar cell; HC, horizontal cell; PR, photoreceptor; OPL, outer plexiform layer; IPL, inner plexiform layer; ONL, outer nuclear layer; INL, inner nuclear layer; RGCL, retinal ganglion cell layer; CMZ, ciliary marginal zone.
