As there is no detectable morphological cycling in either the

Clk knockdown or the DAPT Mef2 rescue ( Figure 5D), Clk is upstream of Mef2 and cycling CLK/CYC activity is important for the circadian regulation of neuronal morphology. Although the reported circadian fasciculation-defasciculation cycle of adult Drosophila s-LNv neurons ( Fernández et al., 2008) had no known molecular connection to the core clock, we report here that the cycle requires the transcription factor Mef2. Mef2 is a direct target of the CLK/CYC complex, which is probably related to the observed mRNA and protein oscillations of Mef2 within PDF cells. Because the fasciculation phenotype of a Clk knockdown is rescued by Mef2 overexpression, it may function as the principal target of the CLK/CYC complex affecting neuronal morphology.

Mef2 itself targets numerous genes affecting neuronal development and morphology, including Fas2. This gene is genetically epistatic to Mef2, as increasing Fas2 levels rescues Mef2 overexpression effects on behavior as well as neuronal morphology. The results indicate that the transcription factor Mef2 links the CLK/CYC complex to Fas2, to circadian alterations in neuronal morphology, and even to locomotor activity rhythms. The mammalian Mef2 family is known to translate extra- and intracellular signals into transcriptional activity in multiple cell types and Kinase Inhibitor Library mw tissues of different species (Potthoff and Olson, 2007). This role is achieved via diverse mechanisms, which include transcriptional, translational, and posttranslational mechanisms as well as collaboration with specific coregulators (Black et al., 1998, Molkentin and Olson, 1996, Nojima et al., 2008 and Sandmann et al., 2007). Neuronal processes are regulated by Mef2, and it also regulates stimulus-dependent changes in synapse number (Flavell et al., 2006). In addition, mammalian Mef2 often plays opposing roles in the regulation of neuronal plasticity. For example, it promotes synapse development during early neuronal differentiation (Li et al., 2008) and then restricts synaptic number at later stages of development (Barbosa et al., 2008). It has similar

dual effects on dendritogenesis, affecting it positively via the miR379–miR410 cluster (Fiore et al., 2009) and negatively in response to cocaine (Pulipparacharuvil et al., 2008). This is likely due to the regulation of different gene Endonuclease sets at different times of development. Despite this complexity, it is possible that Mef2 plays a simple “linear” role in the described cycling of Drosophila PDF neuron fasciculation: the core clock cyclically regulates Mef2 expression, and Mef2 then cyclically regulates, either positively or negatively (such as in the case of Fas2), the transcription of genes functioning in neuronal remodeling ( Figure 6). Relevant to this model are recent experiments in Drosophila by Blau and coworkers, demonstrating cycling Mef2 levels within s-LNv neurons ( Blanchard et al., 2010).