Newly generated Gata2b mutant does not show defects in HSPC generation. (A) Schematic representation of the CRISPR strategy targeting exon 3 of gata2b and the 28-nt integration in gata2b mutants. (B) Alignment of sequencing data of WT gata2b exon 3, where the location of the guide is indicated by the blue arrow at the top of the sequence, and sequencing data from gata2b^−/− DNA showing a 28-nucleotide integration. (C) Gel picture showing genotyping polymerase chain reaction of founder 3 and the F1 with a 28-bp integration in embryos 7 and 8. (D) Gata2b mutation leading to a STOP codon abrogating the protein before the DNA and protein binding zinc fingers. (E) Representative image of ISH for cmyb expression in WT and gata2b^−/− embryos at 26 hours postfertilization (hpf). Bar indicates 100 μm (F) Quantitation of cmyb signal intensity relative to background in WT and gata2b^−/− embryos at 26 hpf. (G) Representative image of runx1 expression in WT and gata2b^−/− embryos at 26 hpf. (H) Quantitation of runx1 signal intensity relative to background in WT and gata2b^−/− embryos at 26 hpf. (I) Example of endothelial-to-hematopoietic transition (EHT) event from WT Tg(fli1a:eGFP) transgenic zebrafish. Time indicated at the bottom right corner in minutes. Scale bar represents 10 μm. Arrow indicates endothelial cell undergoing hematopoietic transition. (J) Quantitation of EHT events between 32 and 40 hpf in WT, gata2b^+/−, and gata2b^−/− embryos. (K) Representative example of runx1 expression in WT and gata2^i4/i4  embryos at 26 hpf in the aorta-gonad-mesonephros region. (L) Quantitation of signal intensity relative to background cells in WT and gata2a^i4/i4  embryos at 26 hpf, where each dot represents 1 embryo (41.4 ± 4.8 and 23.5 ± 2.0; WT, n = 13; gata2a^i4/i4 , n = 21). Error bars represent standard error of the mean. ***P < .001. DA, dorsal aorta; PVC, posterior cardinal vein; sp, somite pair.