dDAAM impairs axonal development in the MB. A, Top, Schematic organization of the dDAAM loci and exon-intron structure of a dDAAM cDNA. The P-element insertion lines, P(GawB)NP6041 and EP(1)1542, were used to create the dDAAM^Ex4 and dDAAM^Ex1 excision alleles, respectively. B, Anti-dDAAM staining (green) of a wild-type, dDAAM^Ex1 and dDAAM^Ex4 mutant brain of young adults. There is weak staining in dDAAM^Ex1 and almost complete lack of staining in dDAAM^Ex4, verifying antibody specificity. C, A Western blot analysis is shown with adult brain extracts from dDAAM^Ex4 and dDAAM^Ex1 mutants. The dDAAM protein can be barely detected in dDAAM^Ex4, and it shows a highly reduced level in dDAAM^Ex1. The 130 kDa band corresponds to the dDAAM protein, whereas the 100 kDa band represents the loading control (α-glycogen phosphorylase). D, The different type of axonal projection defects exhibited by the α and β lobes of dDAAM mutant MBs. FasII staining (green) is used to label the lobes; a wild-type control on the left shows the dorsally running α lobe and the medially projecting β lobe. E, Quantification of the axonal growth and guidance defects exhibited by dDAAM mutants. Data are provided separately for the α and β lobes. Axonal defects can be almost fully rescued by expressing a UAS-FLDAAM construct with the pan-neuronal elav-Gal4 driver line. ***p ≤ 0.0001 (Fisher's exact test). F–I′, Confocal images of GFP-labeled α/β single-cell clones: green represents GFP; red represents FasII. A control single cell clone (F, F′) induced in a wild-type background shows the bifurcated axon with a dorsally and a medially running branch. G, G′, A dDAAM-null mutant clone, induced in a dDAAM^Ex68/+ heterozygous background, also exhibit a wild-type axon morphology. On the contrary, MARCM clones induced in dDAAM^Ex4 homozygous mutants often exhibit guidance defects, most typically when both branches run in the same lobe (H, H′). In other cases, a growth defect is evident when the axon branches are shorter than in wild-type and fail to reach the normal target area (I, I′). The guidance and growth defects sometimes combine, such as in H, H′, where both branches fasciculate into the dorsal lobe but one of them terminates earlier than the other (H′). The α lobe in H is thicker than in wild-type; and concomitantly, the β lobe is thinner than normal. J, K, Compared with wild-type control neuroblast (NB) clones (J), NB clones induced in a heterozygous dDAAM^Ex68/+ mutant background and marked with mCD8::GFP (green) display an aberrant axonal morphology (note the reduced number of β axons in K) in 15% of the cases. L, Quantification of the MB mutant phenotypes observed in dDAAM^Ex68 mutant MARCM clones.