In order to understand how the microtubule cytoskeleton is organized in the branches of class IV dendritic arborization (da) neurons, we analyzed the dynamics of EB1-GFP comets throughout the entire dendritic arbor in vivo. We expressed UAS-EB1-GFP using the class IV specific promoter, ppk-Gal4 and focused on third-instar larvae

96 hr after egg laying, because although Selleckchem IWR-1 the arbor is well established and the primary branches are stable, the terminal branches are still dynamic ( Lee et al., 2011; Parrish et al., 2009; Ye et al., 2007). Using EB1-GFP to mark the growing plus ends of microtubules, we found that microtubules grew predominantly in the retrograde direction toward the cell body in long (>50 μm) primary branches, consistent with previous reports in other classes of neurons ( Figure 1A; Mattie

et al., 2010; Rolls et al., 2007; Satoh et al., 2008; Song et al., 2012; Stone et al., 2008; Zheng et al., 2008). However, in shorter branches (20–30 μm), we detected mixed microtubule polarity ( Figure 1B), similar to that defined in mammalian neurons ( Baas et al., 1988; Kapitein et al., 2010). Branches of this length corresponded to higher order branches, such as the secondary and tertiary branches, from which terminal branches originate (see Figure S1 available online). In even shorter terminal branches (<20 μm), EB1 comets grew predominantly in the anterograde direction toward the distal tip of the branch ( Figures 1C and 1D). Therefore, microtubule orientation within the dendritic arbor correlates with the length of the dendrite branch. Longer, more established SB203580 branches contain predominately retrograde EB1 comets and shorter branches are composed of mainly anterograde EB1 comets not ( Figure 1E). This held true for branches in both the proximal and distal regions of the arbor. The speeds of the anterograde

and retrograde comets were comparable in all branches, and closely matched the growth rates of microtubules in other systems ( Figure 1F; Akhmanova et al., 2001; Stepanova et al., 2003). In order to generate different patterns of microtubule polarity throughout the dendritic arbor, there likely exist a variety of mechanisms for microtubule nucleation. We therefore wanted to understand the origins of anterograde and retrograde EB1 comets growing specifically within the terminal branches. Anterograde comets originated from three main sources: the parent branch (Figure 2A), the branchpoint (Figure 2B), and within the terminal branch (Figure 2C). EB1 comets growing retrogradely along the parent branch could be directed into a smaller daughter branch and grow anterogradely toward the distal tip; however, this was the least common source of EB1 comets for the terminal branches (20% and 5% of anterograde comets in <10 μm and >10 μm branches, respectively) (Figures 2A, 2D, and 2E).