Third instar larvae at 96 hr AEL (unless specified otherwise) were mounted in halo carbon oil and confocal images of class IV da dendrites were collected with a Leica SP5 laser scanning microscope. For high-resolution imaging on the z axis, the larvae were lightly anesthetized with isoflurane JAK inhibitor before mounting. Image stacks with a z step size between 0.16–0.2 μm were acquired with a 40× 1.25 NA oil lens. For quantification

of dendritic phenotypes, eight to ten image stacks were collected from class IV da neurons in A2–A3 segments for every genotype. For short-term time-lapse imaging of dendritic dynamics, the larvae were mounted in a imaging chamber constructed with a thin aluminum slide with a hole in the middle. The bottom of the hole was covered with an oxygen-permeable membrane (model 5793; YSI). The larvae were mounted on the membrane in halo carbon oil. Confocal image stacks were deconvoluted with Autoquant (MediaCybernetics) and analyzed in Imaris (Bitplane). Detailed methods for image analysis and quantification

are described in Supplemental Experimental Procedures. Antibodies used in this study are mouse anti-Mys (1:50, CF.6G11, DSHB), mouse anti-Mew Tyrosine Kinase Inhibitor Library (1:10, DK.1A4, DSHB), rabbit anti-DsRed (1:200, Clontech). Secondary antibodies conjugated to DyLight dyes (Jackson ImmunoResearch) were used at 1:400 dilution. Immunostaining of Drosophila larvae was performed as described ( Grueber et al., 2002). Briefly, third not instar larvae were dissected in cold PBS, fixed in 4% formaldehyde/PBS for 20 min at room temperature (RT), and stained with the proper primary antibodies and subsequent secondary antibodies, each for 2 hr at room temperature. Detailed methods for TEM are described in Supplemental Experimental Procedures. We thank Wes Gruber for communicating

results prior to publication. We thank members of the Jan lab for discussion; Chung-hui Yang for help in cloning and making transgenic lines; Yang Xiang for testing of electrophysiological experiments; Sandra Barbel for help in dendrite tracing; Mark Krasnow, Frieder Schoeck, Jian Wang, Bloomington Stock Center, VDRC, and FlyTrap for fly stocks; DSHB for antibodies. This work was supported by a postdoctoral fellowship from the Jane Coffin Childs Memorial Fund to C.H., a California Institute for Regenerative Medicine (CIRM) grant to S.Z., NIH grant (2R01 GM063891), American Cancer Society (RSG-07-051), and the Knowledge Innovation Program of the Chinese Academy of Sciences KSCX2-YW-R-263 to X.L., and by NIH grant (2R37NS040929) to Y.N.J. L.Y.J. and Y.N.J. are investigators of Howard Hughes Medical Institute. “
“For many types of neurons, dendrites represent the most expansive membrane compartment, with large surface areas in extensive contact with the surfaces of other neurons as well as the substrates upon which they grow.