Fig. 3
The “bushy” phenotype of CASK-mutant neurite arbors in vitro. a The CASK-mutant larval CNS is reduced in size. Stereomicroscopic view of freshly dissected, unfixed whole-CNS explants from wandering third-instar female larvae in buffered saline, dorsal side up, anterior to the top. CASK control (Ex33/Ex33; left) alongside mutant (∆18/∆18; right). OL, optic lobe; Br, brain; *, subesophageal zone; VG, ventral ganglia; pn, peripheral nerves. Scale bar = 100 µm. b-h Photomicrographs of cultured larval CNS neuron, immunofluorescently labeled for a neuronal membrane marker, after 3 div (60X magnification). b NeuronMetrics software output. The color-coded lines have been thickened to improve visibility: yellow polygon, territory; blue, central portion of the neuronal cell body; green, branches of the skeletonized neurite arbor. Scale bar = 10 µm. c-h Scale bar = 20 μm. Neurons representing the ~ 25th (c, f), ~ 50th (d, g), and ~ 75th (e, h) percentiles for each of three arbor-size parameters, total neurite length, territory area, and branch density. c-e CASK control, Ex33/Ex33. f–h CASK mutant, ∆18/∆18. (i-l) The “bushy” phenotype: CASK-mutant neurite arbors are reduced in size with excessive branch density. Quantification of neurite-arbor morphology, depicted as box-plot distributions, comparing 3 div cultures of CASK-control (Ex33/Ex33; n = 106 neurons; aqua) and CASK-mutant (Δ18/∆18; n = 105 neurons; magenta) larval CNS neurons. Center lines and arrowheads represent the 50th percentile. Top and bottom of each box represent the 75th and 25th percentiles, respectively. The upper and lower whiskers represent the 90th and 10th percentiles, respectively. Significance levels: ***, p < 0.0005; ****, p < 0.00005. Total neurite length (i), territory area (j), and higher-order branch number (k) were all reduced in CASK-mutant neurons, whereas branch density (l) was increased. These data are from one of 9 independent experiments; see Additional file: Table A3 for analysis across all experiments