Additional file 12: of Zebrafish prdm12b acts independently of nkx6.1 repression to promote eng1b expression in the neural tube p1 domain

Generation of germ line nkx6.1 mutants. a. Schematic showing genomic sequence of nkx6.1 with the homeodomain indicated in green. The CRISPR target sequence is shown in red with the AvaII restriction site bracketed and the black arrow indicating the AvaII cut site. b. Identification of functional guide RNAs. sgRNA and cas9 mRNA was injected into 1-cell stage embryos. Injected embryos were raised to 24hpf and AvaII digestion of PCR amplicons from pooled embryos was used to identify CRISPR-induced mutations (black arrow). c. Identification of individual F0 founders. sgRNA/cas9 injected embryos were raised to adulthood and crossed to wildtype fish. AvaII digests of PCR amplicons from pools of embryos was used to identify F0 mosaic founders (black arrow). d. Identification of F1 animals. Adult F0 mosaic founders were out-crossed to wildtype fish and the F1 offspring raised to adulthood. AvaII digests of PCR amplicons from fin clip genomic DNA was used to identify heterozygous F1 animals. e. Sequencing of F1 genomic DNA revealed the transmission of two mutant alleles (um321, um322). um321 carries a 23 base pair deletion (black dashes) while um322 carries a 1 base pair insertion (green) and 3 base pair substitutions (blue). The CRISPR target sequence is shown in red. f. Predicted amino acid sequence of mutant alleles. The um320 and um321 peptides share their first 44 amino acids with the wildtype sequence before going out of frame and terminating at a premature stop codon N-terminal to the conserved homeodomain. g. Quantification of the size (along the dorsoventral axis) of the nkx6.1 expression domain in prdm12b MO-injected embryos (data from Fig. 8). (PDF 651 kb)