Fig. 2: Morphology of newly identified s-CPDN₃ and DN_1p subtypes.

A Schematic cycling of Period (PER) and Vrille (VRI) protein abundance in clock neurons¹⁰¹. B tim-(UAS)-Gal4 drives GFP expression in all DN₃, many of which coexpress PER and about 12-19 coexpress VRI. When VRI staining is strong, PER staining is weak or not detectable. Image based on a female brain fixed at ZT1. Representative images are based on expression analyzed in 5 male and 5 female brains. Scale bar = 10 μm (C) DN₃ are closely associated with the lateral horn. D The number of DN₃ per hemisphere accounts for s-CPDN₃, l-CPDN₃, and APDN₃ identified in the connectome. On average, females have slightly more DN₃ (83 ± 5 standard deviation) compared to males (76 ± 6 standard deviation), which could be attributed to DN₃ being more densely packed (and thus difficult to quantify) in males. n = 10 hemispheres from 5 brains. Error bars depict standard deviation. E–I s-CPDN₃ cell types identified in the FlyWire dataset. Numbers in brackets represent the number of neurons in the total brain. s-CPDN₃ could be subdivided into five subtypes. Each subtype comprises two to eight different cell types that have unique morphological characteristics. J Clk4.1M-Gal4 reliably drives GFP expression in 14-15 DN_1p per hemisphere. K Multi-color flip-out (MCFO) analysis reveals previously characterized DN_1pA (4-5 neurons/hemisphere) and DN_1pB (2 neurons) subtypes. MCFO analysis additionally reveals the morphology of uncharacterized DN_1p subtypes. DN_1pC and DN_1pE comprise two cells each per hemisphere L, N. DN_1pD comprises four cells per hemisphere two of which project over the midline while the other two do not, suggesting that the DN_1pD is comprised of two subtypes M. Scale bar = 50μm. Source data for panel D are provided in the Source Data file. Brain mesh is from Dorkenwald et al., 2024.