Adult E cells are labeled by the cry13-Gal4 driver in combination with a Pdf-Gal80 transgene and, along with LNvs, are required to generate normal behavioral rhythms in 12 hr light:12 hr

dark (LD) cycles ( Stoleru et al., 2004). We found that this driver combination only labeled the two larval DN1s ( Figure 1A and data not shown). Although expression of green fluorescent protein (GFP) was often difficult to detect simultaneously in both larval DN1s (as in Figure 1A), expression of UAS-Diphtheria toxin (UAS-Dti) always ablated both larval DN1s, whereas the PDF+ LNvs, the 5th PDF− LNv, and the two DN2s were still present, as judged by clock protein staining (data not shown). This is consistent with larval DN1s becoming the adult DN1a neurons, a subset of adult E cells ( Grima Z-VAD-FMK mouse et al., 2004 and Stoleru et al., 2004). GFP-labeled DN1 projections terminate in the vicinity of the PDF+ UMI-77 molecular weight LNv axonal termini (Figure 1A). Because the GFP derivative used is a postsynaptic marker (Dscam17.1-GFP; Wang et al., 2004), larval DN1 projections

could receive inputs in this region, including from LNvs. To localize DN1 presynaptic termini, we used UAS-Synaptotagmin-HA (UAS-Syt-HA; Robinson et al., 2002) expressed via the stronger cry16-Gal4 driver in combination with Pdf-Gal80 because cry13-Gal4 expression of Syt-HA was undetectable. The two larval DN1s marked by CD8-GFP expression project to the LNv termini in which Syt-HA is detectable in several foci, some of which are very close to LNv axons ( Figures 1B and 1C). Thus, DN1s could signal to LNvs and receive their inputs. This is consistent with electron microscopy studies of adult small ventral lateral neurons (s-LNvs) that

revealed input synapses to s-LNv projections in the dorsal protocerebrum, the Chlormezanone location of adult DNs ( Yasuyama and Meinertzhagen, 2010). We also detected low levels of CD8-GFP and Syt-HA expression in LNvs when expressed with the cry16-Gal4; Pdf-Gal80 combination, presumably because cry16-Gal4 is not completely repressed by Pdf-Gal80. Because cry16-Gal4 also labels a few nonclock neurons in the brain (data not shown), we did not use cry16-Gal4 in the subsequent behavioral experiments. Given the possibility that DN1s signal to LNvs, we first characterized the contributions of these different groups of clock neurons to light avoidance in larvae raised in 12:12 LD cycles at 25°C. In this assay, 15 larvae are placed on a half-covered Petri dish, and the number of larvae on the dark side are counted after 15 min. At 750 lux, ∼70% of wild-type larvae are in the dark at the end of the assay, and this requires the clock genes period (per) and timeless (tim) ( Gong, 2009, Keene et al., 2011 and Mazzoni et al., 2005).