(A–I) Axonal branch pattern at different pupal stages shows excessive branching at early to mid-pupal development. Successive refinement of exuberant branches can be observed between 60 hr and 96 hr (arrowhead, compare D–H). Branch morphology at (A) 36 hr APF, (B and B′) 48 hr APF, (C) 54 hr APF, (D) 60 hr APF, (E) 64 hr APF, (F and F′) 72 hr APF, (G) 84 hr APF, (H) 96 hr APF and (I) adult stage. High magnification of branches is shown in B′ and F′. (J–O) Axonal branch pattern at different pupal stages of EGFRDN expressing DCNs shows excessive branching at early to mid-pupal time points similar to wild type. Impaired refinement of exuberant branches can be observed between 60 hr and 96 hr (arrow, compare L–O). Branch morphology at (J) 36 hr APF, (K and K′) 48 hr APF, (L) 60 hr APF, (M and M′) 72 hr APF, (N) 84 hr APF, (O) 96 hr APF. High magnification of branches is shown in K′ and M′. (P) Quantification of branches at the second branch point at 48 hr and 72 hr APF comparing control and EGFRDN using the Skeleton Analysis tool of ImageJ (‘Materials and methods’). EGFR downregulation does not result in increased branches at 48 hr APF compared to control. Significant decrease of developmental branch numbers at 72 hr APF occurs due to refinement in control. No significant decrease in branch number was observed after EGFR downregulation between 48 hr and 72 hr APF. Compared to control more branches persist after EGFR downregulation at 72 hr APF. Control (48 hr APF) 49.33 ± 9.87 (n = 18), control (72 hr APF) 22.75 ± 9.1 (n = 18, p<0.01), EGFRDN (48 hr APF) 45.77 ± 10.96 (n = 16), EGFRDN (72 hr APF) 37.3 ± 3.83 (n = 14) (to control 72 hr APF, p<0.05). Error bars represent SEM. t test. *p<0.05; **p<0.01. The scale bars represent 20 µm except in B′, K′ and M′ with 10 µm. (Q) Schematic representation of the role of EGFR signaling in DCN axonal branch formation.