Confocal stacks were imaged using an Olympus FV1000 confocal microscope equipped with 20 × /0.85 NA and 100 × 1.40 NA oil-immersion objectives.

Phusion DNA polymerase (New England Bio Labs) was used to amplify the e GFP-Lox P-kanamycin cassette from plasmid PL-452 N-e GFP (Addgene) using using primer exon 1 (lowercase) and 23 nt from the 3′ end of the e GFP cassette (uppercase).

The GFP fragment was excised using Fse I and inserted into ) was constructed via recombineering (Venken et al., 2006).

For imaging Alexa Fluor 488 and Cy3, 488 and 543 nm lasers were used, with the 405/488/543 nm dichroic mirror for excitation.

Fluorescence signals were separated by a beam splitter (560 nm long-pass) and recorded on spectral detectors set to 500–530 and 555–655 nm for Alexa Fluor 488 and Cy3, respectively.

Z-series were obtained at 2 μm step size, and Kalman-averaging was not used.

Original Olympus images were saved as 12-bit oib format and processed using FV1000 confocal software to generate maximum intensity projections (Z-projections). These clusters of brain pacemaker neurons communicate via neuropeptides to form a network that determines the pattern of locomotor activity based on environmental inputs (Peschel and Helfrich-Förster, 2011; Taghert and Nitabach, 2012; Yoshii et al., 2012). Pacemaker neurons can be divided into multiple clusters based on their location, size, and neuropeptide expression, including eight pigment dispersing factor (PDF)-expressing small ventral lateral neurons (s LNs; Helfrich-Förster, 2005; Nitabach and Taghert, 2008). Our initial experiments confirm CLK-GFP expression in ∼16 PER-positive early pacemaker neurons of L1 and L2 larvae. To determine when late pacemaker neurons arise during metamorphosis, a is the first clock gene to be expressed and CLK-GFP can be detected with high sensitivity.Antibody staining of adult brain and larval CNSs was performed as previously described (Houl et al., 2008). Larval CNSs and adult brains were dissected and fixed with 3.7% formaldehyde.