Figure 6. Bsh and Ap form a coherent feed-forward loop to activate DIP-β.

(A–D) Bsh Crispr knockout (KO) in postmitotic L4 neurons results in loss of DIP-β expression in the proximal lamina neuropil (arrow) at 3 days after pupa formation (APF). DIP-β expression is detected using an anti-DIP-β antibody. The signal in the distal lamina is from non-lamina neurons, probably LawF. Significantly reduced DIP-β fluorescence intensity is observed in the proximal lamina (75–100% distance, marked by red bar (C, D)). ***p<0.001, unpaired t-test, n=8 brains, each line represents each brain, scale bar, 10 µm. Genotype: 31C06-AD, 34G07-DBD>UAS-Cas9, UAS-Bsh-sgRNAs. (E–I) Bsh Crispr KO in L4 neurons results in a decrease of primary dendrite length and proximal synapse number in postmitotic L4 neurons of 1-day adults. Here and below, white dash lines indicate the lamina neuropil and yellow lines show the boundary between the distal and proximal lamina. The average number of Brp puncta in L4 neurons present within the distal or proximal halves of lamina cartridges. *p<0.05, ***p<0.001, ns = not significant, unpaired t-test, n=100 cartridges, n=5 brains, each dot represents one cartridge, data are presented as mean ± SEM. Genotype: 31C06-AD, 34G07-DBD>UAS-Cas9, UAS-Bsh-sgRNAs, UAS-myrGFP, UAS-RSR, 79C23-S-GS-rst-stop-rst-smFPV5-2a-GAL4. (J–M) Ap RNAi knockdown (KD) in postmitotic L4 neurons results in loss of DIP-β expression in the proximal lamina neuropil (arrow) at 3 days APF. The signal in the distal lamina is from non-lamina neurons, probably LawF. Significantly reduced DIP-β fluorescence intensity is observed in the proximal lamina (75%–100% distance, marked by red bar (L, M)). ***p<0.001, unpaired t-test, n=8 brains, each line represents each brain, scale bar, 10 µm. Genotype: R27G05-Gal4>UAS-ApRNAi. (N–R) Ap-KD in L4 neurons results in an increase of primary dendrite length and proximal synapse number in postmitotic L4 neurons in 1-day adults. The average number of Brp puncta in L4 neurons present within the distal or proximal halves of lamina cartridges. *p<0.05, ***p<0.001, ns = not significant, unpaired t-test, n=100 cartridges, n=5 brains, each dot represents one cartridge, data are presented as mean ± SEM. Genotype: 31C06-AD, 34G07-DBD>UAS-RSR, 79C23-S-GS-rst-stop-rst-smFPV5-2a-GAL4, UAS-Ap-shRNA, UAS-myrGFP. (S) Summary.

Figure 6—figure supplement 1. DIP-β expression is disrupted when knocking down Ap in L4 neurons.

(A–C) Ap-knockdown (KD) (31C06-AD, 34G07-DBD>UAS-RSR, 79C23-S-GS-rst-stop-rst-smFPV5-2a-GAL4, UAS-Ap-shRNA; see Materials and methods for an explanation of this genotype) significantly reduced Ap expression in L4 neurons at 2 days after pupa formation (APF). The white line circle outlines lamina neuron cell bodies. Scale bar, 10 µm. (C) Quantification of Ap signal in the cell bodies of L4 neurons in control (A) and Ap-KD (B). We averaged the Ap signal of three cell bodies from each brain and normalized the Ap signal by setting the highest Ap signal as 100. ***p<0.001, unpaired t-test, n=6 brains, each dot represents each brain, data are presented as mean ± SEM. (D–F’) DIP-β expression is disrupted in L4 neurons in Ap-KD (31C06-AD, 34G07-DBD>UAS-RSR and 79C23-S-GS-rst-stop-rst-smFPV5-2a-GAL4) and UAS-Ap-shRNA. Note that the DIP-β signal in the proximal lamina belongs to L4 in control (arrow, L4), whereas the DIP-β signal in the distal lamina belongs to unknown cells. Scale bar, 10 µm. (E–G) Quantification of DIP-β fluorescence intensity along the long axis of lamina cartridges (see white lines in (D) and (F)) from the distal dash line to the proximal dash line. Significantly reduced fluorescence intensity is observed in the proximal lamina (75–100% distance, marked by the red bar in (E) and (G)) of Ap-KD flies compared to the control. ***p<0.001, unpaired t-test, n=5 brains, each line represents each brain.