Fig. 4: Single neuron morphology and development of H-neurons in NetB pathway loss-of-function.

a, b Single-cell clone analysis of H-neurons in netB^Δ a and 72A10-Gal4 > unc-5-RNAi b flies. Confocal images of representative single neuron types are labelled as in Fig. 1j–m and their occurrence indicated as n numbers. Insets at the bottom show higher magnification of AB region with representative single neuron types (magenta) and the whole 72A10 neuron circuit (yellow). Dotted line represents the brain midline. Scale bars, 30 µm. c Development of the netB^Δ mutant H-circuit during pupal stages. In contrast to wild-type ASYM flies, netB^Δ H-neuron projections remain in their initial symmetrical state (compare with Fig. 2a). Confocal images of representative netB^Δ mutant H-neurons (magenta) stained with anti-Fas2 antibody (cyan) during pupal stage. ABs (white dotted lines) and noduli (white arrows) are shown. Phenotype categories are defined as in Fig. 2a. Scale bars, 10 µm. d Evolution of the netB^Δ mutant H-circuit during pupal development (28–48 h APF) showing that in contrast to wild-type, the decrease of the symmetric phenotype does not reach a plateau as in wild-type flies (compare with Fig. 2b) but instead increases in a second phase, so no occurrence of the “right” phenotype is observed in netB^Δ mutant flies. (n = 21 brains for 28–30 h APF and n = 20 brains for 32–34, 36–38, 40–42, and 44–46 h APF representing the merging of 2 adjacent data points shown in f; n = 10 for 48 h APF). Source data are provided as a Source Data file. e. Summary of symmetrical H-neuron pattern formation in netB^Δ mutant flies during pupal stage. f, g Morphometric analysis of NetB mutant’s H-neurons during pupal development (28–48 h APF), showing the evolution of the volume of left and right AB projections f, and the number of left and right H-neuron cell bodies g. AB projections volume: n = 11 brains for 28 h APF; n = 10 for all other time points. H-neuron cell bodies (L/R): n = 10/10 for 28, 30, 46, and 48 h APF; n = 9/10 for 32 and 38 h APF; n = 10/9 for 34 and 42 h APF; n = 9/9 for 36, 40, and 44 h APF. Data are presented as mean ± standard error of the mean (SEM). h Temporal requirement for netB and unc-5 function during development, showing sequential gene activities taking place before symmetry breaking. Data represent the frequency of adult SYM phenotype following temporally restricted netB and unc-5 loss-of-function. Solid red and green line curves represent local regression of the frequency data obtained with 2 different methods: either ON > OFF/OFF > ON RNAi expression (data plotted as red and green circles) or using 24 h pulse RNAi expression (data plotted as red and green crosses). 20 brains are analysed for each data point and for both methods (see text, source data file and Methods for details). Shaded areas around solid black line curves represent 95% confidence interval. dPF, days post-fertilisation (25 °C); hrs APF, hours after puparium formation (25 °C). Source data are provided as a Source Data file.