A conserved maternal-specific repressive domain in Zelda revealed by Cas9-mediated mutagenesis in Drosophila melanogaster
Fig 1
ZLD-PB is the dominant isoform expressed both in the embryo and imaginal disc.
(A) Schematic of predicted transcripts from the zld locus (above). Boxes indicate exons with coding sequence (light gray) and untranslated regions (dark gray). The length of the resulting mRNA is given in nucleotides (nt). Two gRNAs flanking the downstream exon of zld-RD used to generate an isoform specific deletion are shown. Schematics of the predicted protein products for each splice variant (below) with amino acid numbers in D. melanogaster ZLD shown above. Above are the approximate locations of the transcriptional-activation and DNA-binding domains as demonstrated in Hamm et al (2015) [20]. (B) Confocal images of embryos homozygous for either an N-terminal mCherry-tagged ZLD or for ZLD-PD-mCherry at stages 5, 12–13, and 14–16. The first column shows the maximum projection images for mCherry-ZLD expressing embryos. All other images show a single confocal slice. ZLD-PD-mCherry (1) and (2) indicate embryos from two distinct editing events. (C) Confocal images of mCherry-ZLD isoforms demonstrating the endogenous ZLD and ZLD-PD specific expression in third instar larval wing discs. Outlines show the borders of the wing discs as determined by transmitted light images. All images shown are the maximum projection. (D) Immunoblot for ZLD on S2 extract from cells expressing either ZLD-PB or ZLD-PD or total lysate from third instar wing discs. (E) Sequence of zld-RD following Cas9-mutagenesis demonstrating removal of the splice acceptor and coding sequence. Small insertions (red sequence) and deletions (dashed lines) shown. Lower case letters indicate intron sequence. Capital letters indicate exonic sequence.