Fourth, most of the enclosed dendrites are in the medial-lateral (or dorsal-ventral) orientation. With the exception of the enclosed dendrites, most class IV da dendrites are thus located in a 2D sheet at the interface of the epidermal basal surface and the ECM. We then performed time-lapse analyses of how epidermal cells enclose dendrites by imaging the ventral dendritic field of the same ddaC neurons at 72 hr after egg laying (AEL) (Figure 2A) and 84 hr BMS777607 AEL (Figure 2B) and

comparing the distribution of enclosed dendrites. Newly enclosed dendrites were found to emerge in three different ways. First, stabilized branches initially attached to the ECM can subsequently

become enclosed (arrowheads). Second, an enclosed dendrite tip can continue to grow within the epidermal layer (arrows). Third, a dendrite tip that was attached to the ECM can extend a new segment into the epidermal layer (open arrowheads). How do existing and new dendrites grow into the epidermal layer? The first scenario can result from the basal plasma membrane of epidermal cells wrapping around an existing dendritic branch. The second and third scenarios indicate that dendrite tips can grow inside the epidermal layer either by “burrowing a tunnel” or by pushing through spaces between cells. To test these hypotheses, we further examined the spatial relationship of class IV da dendrites find more and the epidermis by transmission electron microscopy (TEM). In order to unequivocally identify the neuronal structures of interest, we used two pre-embedding staining strategies to specifically label the dendrites of class IV da neurons. The first strategy involved antibody staining against RFP in ppk-CD4-tdTom animals, with subsequent

HRP-conjugated secondary antibody labeling. The second strategy was to express a membrane-tethered HRP transgene, UAS-HRP-DsRed-GPI, in class IV da neurons with an improved ppk-Gal4 that has higher and more specific expression (see Experimental Procedures). In both cases, the HRP reaction product Diaminobenzidine (DAB) can be detected by TEM ( Larsen et al., 2003). Our TEM analysis revealed that, whereas most dendrites are TCL located underneath the basal surface of epidermal cells and are in direct contact with the ECM ( Figures 2D–2F), there are three types of dendrite enclosure in the epidermal layer. First, we observed thick enclosed dendrites connected to the ECM through a channel formed by opposing epidermal cell membranes ( Figure 2G), suggesting the wrapping of existing dendrites by the epidermal basal surface. Second, some dendrites are located between cell junctions of neighboring epidermal cells ( Figure 2H), confirming that dendrites can indeed grow between epidermal cells.