Requirement for chromatin remodelers rbbp4 and hdac1 in zebrafish neurogenesis. (A) CRISPR target site in exon 2 of rbbp4 used to isolate 4 bp frameshift mutation rbbp4Δ4-is60. PAM sequence is underlined. SmlI restriction enzyme site overlapping the target site is shown in red. (B) 5 dpf wild-type (+/+) larva. (C) Gross phenotype of 5 dpf homozygous mutant rbbp4Δ4/Δ4 larva showing microcephaly and microphthalmia. (D) CRISPR target site in exon 5 of hdac1 used to isolate 4 bp frameshift mutation hdac1Δ4-is70. PAM sequence is underlined. (E) 3 dpf wild-type larva. (F) Gross phenotype of 3 dpf homozygous mutant hdac1Δ4/Δ4 larva showing microcephaly and retinal coloboma (arrow). (G) Diagram of 2 dpf larval midbrain and retina. (H-J) Sections of wild-type (H), rbbp4Δ4/Δ4 (I) and hdac1Δ4/Δ4 (J) 2 dpf larval heads labeled with neural differentiation marker HuC/D (red) and apoptosis marker activated caspase 3 (green). In rbbp4Δ4/Δ4, apoptosis is present in the dorsal and lateral tectum (arrows), and throughout the inner retina (brackets) (I). hdac1Δ4/Δ4 larval brain and retina are smaller than wild type, but few apoptotic cells are detected in the brain or retina (J). (K,L) Quantification of activated caspase 3 in the midbrain (K) and retina (L) of 2 dpf wild-type, rbbp4Δ4/Δ4 and hdac1Δ4/Δ4 larvae. OT, optic tectum; Th, thalamus; R, retina. Scale bars: 500 µm (B,C); 200 µm (E,F); 100 µm (H-J).
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