Fig. 2

Fig. 2. Validating her3 frameshift mutations. (A) Schematic of strategy for her3 mRNA expression assay. Zebrafish embryos were co-injected with a mixture of two different mRNAs: one of four different her3-P2A-sfGFP generated from either wildtype her3 or each of the three mutant her3 zebrafish; and a control TagRFPT mRNA. mRNA mixtures were injected into the yolk of zebrafish embryos at the 1–4 ​cell stage (see Fig. S5 for sequences). (B) At 24 ​h post fertilization (hpf), embryos were imaged on a Leica M205FA fluorescent microscope to quantify GFP and RFP fluorescence. GFP fluorescence indicates either the her3 wildtype or her3 mutant P2A-sfGFP construct, whereas TagRFPT is an injection control. Scale bar is 500 ​μm. (C) Plotted is the raw integrated density ratio of GFP to RFP fluorescence. Each point represents an individual fish. Wildtype (WIK): n ​= ​18. nch1: n ​= ​19. nch2: n ​= ​16. nch3: n ​= ​15. A Brown-Forsythe and Welch ANOVA was performed with Dunnett T3 multiple comparison test. The comparisons include wildtype WIK to her3 mutants with the following P values: WIK- her3^nch1: p ​= ​1.20 ​× ​10⁻⁷. WIK- her3^nch2: p ​= ​5.24 ​× ​10⁻⁸. WIK- her3^nch3: p ​= ​1.60 ​× ​10⁻⁷. (D) Western blot of 6hpf embryos from her3^nch1 and her3^nch3 homozygotes, and a compound heterozygote for her3^nch1/nch2. A cross-reactive human HES3 antibody was used to detect zebrafish wildtype Her3, and a Tubulin antibody was used as a loading control.