Fig. 1 - Supplemental 1

Generation of betaPix conditional trap (betaPixct) allele by an HDR-mediated knock-in method. (A) Sanger sequencing results of the original pZwitch (+3) and the modified vector with glycine–serine–glycine spacer (GSG) in front of the 2A peptide. (B) Sanger sequencing results of the genomic PCR products at both junction sites from the betaPixct F1 offspring showing the correct knock-in in the betaPix locus. (C) The screening process for betaPixct knock-in zebrafish showing about 1% efficiency with correct homologous recombinations. (D) Representative stereomicroscopy images of o-dianisidine staining in bbhfn40a embryos at 48 hpf. Lateral view, anterior to the left in left panel; Dorsal view, anterior to the top in right panel. (E) Quantification of hemorrhagic parameters in (D). Left panel showing hemorrhage percentages, with independent experiment as dot. Right panel showing hemorrhage areas with each dot representing one embryo. # represents the numbers of embryos scored for each analysis, three or more individual experiments conducted. Data are presented in mean ± SEM; unpaired Student’s t-test with individual p-values mentioned in the figure. (F) 3D reconstruction of the betaPix expression (magenta) in betaPixct/+ brains of embryos microinjected with PBS or Cre mRNA at 24, 48, 72, and 144 hpf. Cre-induced RFP expression indicates successful inversion of gene trap system (arrows). Dorsal view, anterior to the top. (G) 3D reconstruction of the neurod1 transgenic expression (green) and betaPix expression (magenta) in betaPixct/+ brains of embryos microinjected with PBS or Cre mRNA at 24, 48, 72, and 144 hpf. Cre-induced RFP expression indicates successful inversion of gene trap system (arrows), marking strong betaPix expression at the midbrain hindbrain boundary (MHB), and milder expression at hindbrain at early stages. At 144 hpf, betaPix expresses ubiquitously in the brain, outlining the cerebellum (arrowheads). Dorsal view, anterior to the top. Individual scale bars are indicated in the figure.