Fig. 4

Fig. 4. her3 null mutation results in deregulation of neurog1 expression. (A–B) RNAscope confocal images of her3 (green) and neurog1 (purple) in 11hpf WIK (A) and 11hpf her3^nch1 homozygous (B) embryos. White arrowhead denotes anterior of the embryo. White dotted line outlines the yolk of the embryos. (C–D) Integrated density of the her3 (C) and neurog1 (D) expression are quantified. Both WIK and her3^nch1: n ​= ​3 samples per condition. Statistical significance was determined by a Welch's two-tailed t-test. her3: p ​= ​0.0306. neurog1: p ​= ​0.0470. (E) qRT-PCR for neurog1 and her3 using 11hpf WIK and her3^nch1 embryos. qRT-PCR includes four biological replicates per line, with each replicate containing a pool of 40, 11hpf embryos. Each biological replicate is comprised of three technical replicates. Statistical significance was determined by an unpaired Welch's t-test. neurog1 p ​= ​2.11 ​× ​10⁻⁴. her3 p ​= ​4.14 ​× ​10⁻⁴. (F) ChIP-qPCR using 6hpf WIK embryos injected with FLAG-tagged her3 mRNA cloned from either wildtype WIK zebrafish or her3^nch1 zebrafish. For each mRNA injected, n ​= ​3 biological replicates are shown from independent experiments. For each sample, one million zebrafish cells were combined with one million Rh30 ​cells (human rhabdomyosarcoma cell line) as carrier chromatin. After FLAG ChIP for the wildtype Her3 or Her3^nch1 mutant construct, qPCR was performed targeting known Her3 binding sites in the neurog1 promoter. Fold enrichment was calculated by comparing FLAG tagged Her3 or Her3^nch1 mutant binding in the neurog1 promoter as compared to a gene desert. Statistical significance (p ​= ​4.46 ​× ​10⁻²) was calculated using an unpaired Welch's t-test.