Figure 5. Notch activation and Bsh expression are mutually independent; Notch signaling without Bsh specifies L3 neuron type.

(A–F) Notch activation and Bsh expression are mutually independent. (A–C) N-KD in lamina (27G05-Gal4, tubP-GAL80[ts], UAS-Notch-RNAi) results in loss of Hey expression without affecting Bsh expression. (C) Quantification (single optical section). Here and below, scale bar, 10 µm, n≥5 brains. (D–F) Bsh-KD in lamina (27G05-Gal4>UAS-Bsh-RNAi) results in loss of Bsh expression without affecting Hey expression. (F) Quantification (single optical section). (G–L) Notch signaling without Bsh specifies L3 neuron type. (G) Schematic of the genetics used to trace the morphology of Hey+ lamina neurons. (H, I) In controls, GFP+ neurons express the L4 marker Bsh (white arrowhead) at 19 hr APF. In Bsh-KD, Bsh expression becomes absent in lamina and GFP+ cells now express L3 markers Zfh1 and Erm (white arrowhead). (J–L) In controls, GFP + cells express the L4 marker Ap (white arrowhead) and have L4 morphology (asterisk) at 2.5d APF. In Bsh-KD, GFP+ cells express L3 marker Erm (white arrowhead) and adopt L3 morphology (asterisk). (L) Quantification for (J) and (K) (single optical section). (M–O) (M) Schematic of the genetics used to trace the morphology and presynaptic sites of Hey+ neurons. (N, O) In controls, Tomato+ neurons have L4 morphology and presynaptic sites (Brp) in the lamina. In Bsh-KD, Tomato+ neurons adopt L3 morphology and connectivity, which lacks presynaptic localization in the lamina (Xu et al., 2019). (P) Summary. Data are presented as mean ± SEM. Each dot represents each brain. n=5 brains in (C), (F), and (L). ***p<0.001, ns = not significant, unpaired t-test.