Fig. 3
Generation and expression of the hbnKOGal4 strain. A The genomic organization of the hbn locus is shown with the positions of the five exons [25] relative to the next upstream and downstream genes (CG15649 and CG33704). Noncoding regions are indicated by white boxes, and coding regions are indicated by blue boxes. The region deleted in the gene targeting construct is indicated by black arrowheads. B The genomic organization of the hbnKOGal4 strain is indicated. Here, the region upstream of the ATG in exon 1 up to sequences immediately downstream of the exon 1 donor splice site was deleted and replaced by Gal4 (yellow) flanked by an attP/B site (red) and a loxP site (green). C-E Laser confocal images show the expression in hbnKOGal4 heterozygous animals at different developmental stages visualized using a UAS-mCD8::GFP strain and a UAS-H2B-mRFP1 strain. C In a stage 16 embryonic brain, Hbn expression is shown in red, and hbnKOGal4 dependent marker GFP expression is shown in the membrane in green. Coexpression of GFP and Hbn is observed in several regions of the embryonic brain (white arrowheads), and axons crossing from one brain hemisphere to the other are also labelled (yellow arrowhead). D In the right hemisphere of a third instar larval brain, Hbn and the hbnKOGal4 marker coexpression is observed in the DM lineages, mushroom bodies (MB) and dorsal inner proliferation centre (dIPC). In the medulla (ME), the GFP marker is expressed in some regions alone (D‘) compared to Hbn expression (D‘‘). E In the right part of an adult brain coexpression of Hbn and RFP is detectable in the medulla (ME) and an additional domain between VLP and SLP (yellow arrowhead) (see Fig. 1J for comparison). F In a stage 16 homozygous hbnKOGal4 embryo stained with an anti-HRP antibody, the typical hbn mutant brain phenotype is visible. The size of the supraesophageal brain commissure (SEC) is substantially reduced (white arrowhead), the protocerebral connectives (PCN) are disorganized (yellow arrowhead) and the size of the anterior protocerebrum is reduced (red arrowhead) (G) A stage 16 wild-type embryo is shown for comparison. Abbreviations: DM, dorsomedial lineage; dIPC, dorsal inner proliferation centre; MB, mushroom bodies; ME, medulla. (Scale bars: C, F, G, 25 μm; D-D‘‘, E, 50 μm)