Fig. 3

Fig. 3. her3 null mutation recapitulates her3-morpholino phenotype. (A) qRT-PCR for her3 from each her3 null mutant line compared to the wildtype WIK. qRT-PCR was done with 3 biological replicates per line, with each replicate containing a pool of 10, 24hpf embryos. Each biological replicate is comprised of three technical replicates. Statistical significance was determined by a one way ANOVA with Dunnett multiple comparison test. WIK- her3^nch1: p ​= ​1.82 ​× ​10⁻³. WIK- her3^nch2: p ​< ​1.00 ​× ​10⁻⁴. WIK- her3^nch3: p ​< ​1.00 ​× ​10⁻⁴. (B) qRT-PCR for her3 with WIK, her3-MO, and mismatch (mm)-MO. 5–10 ​nL of morpholinos at a concentration of 500 ​μM were injected into the yolk of WIK embryos at the 1–4 ​cell stage. qPCR was done with 3 biological replicates per condition, with each replicate containing a pool of 10, 24hpf embryos. Each biological replicate is comprised of three technical replicates. Statistical significance was determined by a one way ANOVA with Dunnett multiple comparison test. WIK-her3-MO: p ​< ​1.0 ​× ​10⁻⁴. WIK-mm-MO: p ​= ​8.46 ​× ​10⁻¹, not significant. (C) RNAscope on whole mount 24hpf WIK wildtype zebrafish embryos. RNAscope targeting her3 RNA transcripts (red) was done and counterstained with DAPI (blue). Representative image was taken on a Leica M205FA fluorescent stereoscope out of three embryos evaluated. Scale bar is 500 ​μm. (D–E) RNAscope targeting her3 RNA transcripts was done on 24hpf WIK (D) and 24hpf her3^nch1 (E) embryos and counterstained with DAPI. Images were taken on a Zeiss LSM800 confocal microscope with a 20x objective. Images shown are z-stack projections: (D) WIK is a projection of 53, 2 ​μm slices; (E) her3^nch1 is a projection of 48, 2 ​μm slices. her3 is shown in red and DAPI is shown in blue. (F) Quantification of the area of her3 expression from (D–E) indicated by white arrowheads. WIK and her3^nch1: n ​= ​3 embryos per condition. A Welch's two-tailed t-test had a p-value of 2.62 ​× ​10⁻².