Figure 4.

WT EBF3 and knot Rescue Lethality, but EBF3 p.Arg163Gln and p.Arg163Leu Fail to Rescue Lethality in Flies

(A–D) For expression analysis of third instar brain and ventral nerve cord, yw/y; kn-T2A-GAL4/CyO, Kr-GAL4, UAS-GFP males were crossed with yw; UAS-cDNA/TM3 Sb, Kr-GAL4, UAS-GFP virgin females, and double heterozygotes were selected by loss of GFP expression in the wandering third instar larval stage. Images were acquired on a Leica Sp8 laser-scanning confocal microscope. The same settings for laser power and detector gain were used for all genotypes. Third instar larval brain images were acquired as a z stack with a z-step of 1.51 μm and line average of 4 at 400 Hz with a 20× objective at 1024 × 1024 pixel resolution. Maximum intensity projections were created from the stack in ImageJ. Immunolabeling revealed that kn-T2A-GAL4 drives expression of HA-tagged UAS fly lines for WT Knot (A), WT EBF3 (B), EBF3 p.Arg163Gln (C), and EBF3 p.Arg163Leu (D) in the third instar brain and ventral nerve cord (pan-neuronal marker Elav in cyan; nuclei labeled with DAPI in gray). The scale bar represents 100 μm.

(E) Fly in vivo rescue analysis using the UAS-GAL4 system. For generating the rescue flies, w¹¹¹⁸/y; Df(2R)BSC429/Sp; UAS-cDNA-WT-HA/+ males were crossed with yw; kn-T2A-GAL4/SM6a virgin females to produce rescue animals with Knot or EBF3 produced solely from the UAS allele under the control of the kn-T2A-GAL4 driver. The genotypes of the rescued flies are yw/y; Df(2R)BSC429/kn-T2A-GAL4; UAS-cDNA-HA/+ males and w¹¹¹⁸/yw; ; Df(2R)BSC429/kn-T2A-GAL4; UAS-cDNA-HA/+ females. For each UAS-cDNA line, >550 adult flies were scored; data represent the number of observed rescue flies and the total number of flies scored. UAS fly lines expressing WT Knot or WT EBF3 rescued embryonic lethality in viable adults. UAS fly lines expressing EBF3 variant p.Arg163Gln or p.Arg163Leu completely failed to rescue the lethality such that no rescue animals were observed as adults or pupae.