, 2010 and McDonald and Rosbash, 2001). Taken together with the data showing that Mef2 is a direct target of the CLK/CYC complex ( Figure 5A), the mRNA enrichment and restricted cycling suggest that CLK binding to the Mef2 promoter is spatially limited and includes PDF neurons. Mef2 is also important for the activity-dependent plasticity of s-LNv neuron morphology (Figure 2A). It is notable that the effect of firing on s-LNv morphology fits with the reported increase of s-LNv electrical activity around lights-on (Cao and Nitabach, 2008); this is when the open

conformation of the s-LNv dorsal projections is normally observed. click here Although neuronal firing may affect core circadian oscillator function to influence these circadian morphological changes, we prefer the interpretation that it acts primarily downstream to influence Mef2 transcriptional activity and possibly Mef2 levels as shown in mammalian and amphibian experiments (Chen et al., 2012 and Cole et al., 2012). Alternatively, firing may modulate Mef2 activity via posttranslational modification

(Flavell et al., 2006 and Shalizi et al., 2006). To identify Mef2 target genes, we performed check details ChIP-Chip analysis on fly head chromatin. Mef2 binding undergoes circadian cycling, and among its top targets are genes relevant to neuronal function, axonal fasciculation, and cell adhesion. These include the gene encoding the NCAM homolog Fas2 as well as genes implicated in various aspects of axonal cytoskeleton dynamics, which influence both actin (e.g., Ptp61F, fray, sif, Sema-1A, and the Profilin homolog chickadee)

and microtubules of (Fmr1 and tau). Like Mef2, Fas2 and some other genes involved in cytoskeletal dynamics have cycling mRNAs in purified Drosophila PDF neuron RNA but not in whole-head RNA ( Kula-Eversole et al., 2010 and Nagoshi et al., 2010). The contribution of axonal fasciculation to circadian changes in s-LNv morphology was originally proposed (Fernández et al., 2008) based in part on the circadian regulation of cell adhesion molecules in adult Drosophila ( Ceriani et al., 2002 and McDonald and Rosbash, 2001). However, it is possible that the circadian morphological changes of PDF axons reflect additional mechanisms, including changes in axonal sprouting and retraction as well as fasciculation. The extreme truncated phenotype of Fas2 overexpression makes some contribution from sprouting retraction likely. In any case, Fas2 overexpression clearly rescues the Mef2 overexpression phenotype ( Figure 3). We interpret the failure of Fas2 overexpression to allow circadian morphological changes in an otherwise wild-type background to be due to excess Fas2. Mef2 overexpression should reduce endogenous Fas2 levels, which may bring overall Fas2 into a biologically acceptable range.