Mop3 is an essential component of the master circadian pacemaker in mammals

Abstract

Circadian oscillations in mammalian physiology and behavior are regulated by an endogenous biological clock. Here we show that loss of the PAS protein MOP3 (also known as BMAL1) in mice results in immediate and complete loss of circadian rhythmicity in constant darkness. Additionally, locomotor activity in light-dark (LD) cycles is impaired and activity levels are reduced in Mop3-/- mice. Analysis of Period gene expression in the suprachiasmatic nucleus (SCN) indicates that these behavioral phenotypes arise from loss of circadian function at the molecular level. These results provide genetic evidence that MOP3 is the bona fide heterodimeric partner of mCLOCK. Furthermore, these data demonstrate that MOP3 is a nonredundant and essential component of the circadian pacemaker in mammals.

Figure 1. Generation of mMop3^−/− Mice

(A) Schematic diagram of the region surrounding the bHLH domain of mMop3, the targeting construct, and the resulting mutant allele. Exon numbers reflect known coding exons with exon one being the furthest 5' known start site for MOP3 protein. Dashed lines indicate the regions of homology used for homologous recombination. Solid lines indicate fragment sizes detected by probe (P) following XbaI (X) digest of genomic DNA. Dotted lines represent the fragment sizes generated by PCR genotyping of wild-type and mutant alleles. (B) Southern blot of mouse tail biopsies showing bands of 4.8 kb and 3.2 kb indicating the presence of the wild-type and mutant alleles, respectively. (C) PCR genotyping of tail biopsies showing bands of 400 bp and 600 bp indicating the presence of the wild-type and mutant alleles. (D) Northern blot of mRNA extracted from whole brain tissue. (E) Schematic of the predicted amino acid sequence of isolated transcript from the Mop3 mutant allele. Black arrows show position of RT-PCR primers and a, b, and c correpond to the three isolated fragments. All three fragments corresponded to a single splice variant that would result in the truncation of the protein 15 amino acids after the splice junction between exon-3 and NeoR sequence (vertical bar).

Figure 2

Wheel-Running Activity in Wild-Type and Mop3^−/− Mice (A–D) Representative activity records of individual wild-type (A and B) and Mop3^−/− (C and D) littermates are presented in double-plotted format. The bar above the activity record shows the light–dark cycle. As indicated to the right of each record, animals were individually housed in a light–dark (LD) cycle for 14 days and then transferred constant darkness (DD). On day 22 in DD, animals received a 6 hr light pulse (300 lux) given at CT16 (indicated by the arrow). 10 days after the light pulse, animals were returned to LD for 18 days and then released into constant darkness for 3 days.

Figure 3. mPer1 and mPer2 Expression in the SCN of Wild-Type and Mop3^−/− Mice

Coronal brain sections from mice sacrificed every 4 hr from 54 hr to 82 hr in constant darkness were hybridized with mPer1 and mPer2 riboprobes. (A–E) mPer1 in situ hybridization. (A) Wild-type mPer1 hybridization at 66 hr, which corresponds to CT6; (B) wild-type mPer1 hybridization at 78 hr, or CT18; (C) Mop3^−/− mPer1 hybridization at 66 hr; (D) Mop3^−/− mPer1 hybridization at 78 hr; (E) time course of mPer1 expression in the SCN of wild-type (filled circles) and Mop3^−/− (open circles) mice. (F–J) mPer2 in situ hybridization. (F) Wild-type mPer2 hybridization at 70 hr, which corresponds to CT10; (G) wild-type mPer2 hybridization at 82 hr, or CT22; (H) Mop3^−/− mPer2 hybridization at 70 hr; (I) Mop3^−/− mPer2 hybridization at 82 hr; (J) time course of mPer2 expression in the SCN of wild-type (filled circles) and Mop3^−/− (open circles) mice. The bar at the top indicates subjective night in black and subjective day in gray. Asterisks indicate significant differences between wild-type and Mop3^−/− mice at the times shown (mPer1 GLM ANOVA, F(7,35) 16.41, p < 1.0 × 10⁻⁶; mPer2 GLM ANOVA, F(7,35) 18.61, p < 1.0 × 10⁻⁶, Tukey-Kramer posthoc comparison p < 0.05).

Figure 4. Analysis of mDbp, mMop3, and mMop9 mRNA in Peripheral Tissues of Mop3^−/− Mice

Analysis of mRNA levels analyzed using TaqMan technology™ from livers taken over a time course of 54–82 hr in constant darkness from wild-type animals (filled circles) and null animals (open circles). The bar at the top indicates subjective night in black and subjective day in gray. (A) Expression of mDbp. Asterisks indicate significant differences between wild-type and Mop3^−/− mice at the times shown (GLM ANOVA, F(7,34) = 9.36, p = 5.0 × 10⁻⁵, Tukey-Kramer posthoc comparison p ≤0.05). (B) Expression of mMop3. Asterisks indicate significant differences between wild-type and Mop3^−/− mice at the times shown (GLM ANOVA, F(7,34) 3.52, p 0.012, Tukey-Kramer posthoc comparison p < 0.05). (C) Expression of mMop9. Asterisks indicate significant differences between wild-type and Mop3^−/− mice at the times shown (GLM ANOVA, F(7,34) 2.75, p 0.036, Tukey-Kramer posthoc comparison p < 0.05).