Figure 3.

Mef2 expression in LN_vs is regulated by light and by clock genes. A, B, Representative images (A) and quantification (B) of Mef2 levels in adult s-LN_vs. y w control brains were stained with antibodies to Mef2 (green) and PDF (blue) at ZT9 or ZT21 in LD and CT9 or CT21 on the second day of DD. Clk^ar, cyc⁰, per⁰ and tim⁰¹ brains were also stained with antibodies to Mef2 at ZT9 and ZT21. The clock mutant images are from ZT21. A Pdf-RFP transgene was included to help mark LN_vs in clock mutants and the red channel was used to record RFP fluorescence since endogenous PDF levels are low in Clk^ar and cyc⁰ mutants. The strongly Mef2-staining cells visible in cyc⁰ mutants are not LN_vs. Mef2 levels in adult s-LN_vs were higher at ZT21 than at ZT9 (p < 0.0001) but not different between CT9 and CT21 (p = 0.42). Mef2 levels at ZT21 are higher in per⁰ and tim⁰¹ mutants than in Clk^ar and cyc⁰ mutants (p < 0.01). C, D, Representative images (C) and quantification (D) of Mef2 levels in larval LNs. y w (control) and clock mutant brains were stained as in A. The clock mutant images are from ZT21. The strongly Mef2+ cells visible in cyc⁰ mutants are not LN_vs. Mef2 levels at ZT21 are higher than ZT3 (p < 0.01). Mef2 levels are higher in per⁰ and tim⁰¹ mutants than in Clk^ar and cyc⁰ mutants (p < 0.002) and show time-dependency in per⁰ and tim⁰¹ mutants (p < 0.05). E, F, Representative images (E) and quantification (F) of Mef2 levels in larval LNs at ZT21. tim(UAS)-Gal4/Mef2^EP1751 had higher Mef2 levels than Mef2^EP1751/+ controls (Con, p < 0.05). tim(UAS)-Gal4/+; UAS-Mef2^RNAi/ UAS-Dcr-2 larvae had lower Mef2 levels than UAS-Mef2^RNAi/+ control larvae (p < 0.0005).