1. The Circadian Clock Is a Key Driver of Steroid Hormone Production in Drosophila 
Francesca Di Cara, Kirst King-Jones 
Current Biology 
Volume 26, Issue 18, Pages (September 2016)
DOI: /j.cub
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2. Current Biology 2016 26, 2469-2477DOI: (10.1016/j.cub.2016.07.004)
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3. Figure 1 Functional Characterization of the PG Clock
(A) timeless gene structure (RL mRNA isoform). White indicates coding exons, and gray indicates non-coding exons. Lines indicate introns. Triangle indicates the position of the stop codon in the tim01 null allele. Arrows indicate dsRNA coverage of RNAi transgenes. VDRC, Vienna Drosophila Research Center RNAi line no.  TRiP, Transgenic RNAi Project line no
(B) Phenotypes caused by PG-specific tim-RNAi. Control (left) shows maximal larval size in controls. w1118, genetic background of RNAi lines. PG>, PG-specific expression of Gal4.
(C) D. pseudoobscura fosmid rescues PG>tim1-RNAi animals to pupal and adult stages. Control: pupa shown for size comparison. An independent transgene, UAS-GFP, caused no rescue.
(D) Quantification of D. pseudoobscura fosmid rescue. Error bars represent SD.
(E) PG>RNAi of circadian genes caused larval and pupal lethality (see Table S1 for details).
(F) Immunostaining of TIM and PER in the PG of age-matched L3 larvae (4–6 hr after L2/L3 molt). ZT2, beginning of photophase; ZT18, late scotophase. Scale bars, 30 μm. Dotted rectangle: enlarged in ZT18∗. Arrowhead indicates non-specific stain often seen with this antibody. WT, wild-type.
(G) qPCR profile of tim in PGs during a 24 hr cycle in age-matched L3 larvae (4–6 hr after L2/L3 molt). Error bars represent 95% confidence intervals.
See also Figures S1 and S2 and Tables S1 and S2.
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4. Figure 2
Transcript Profiling Reveals Ecdysone Defects in tim1- and per-RNAi Animals
We used RNA-seq on hand-dissected RGs (which contain the PG) to examine the effects of PG-specific tim1-RNAi and per-RNAi.
(A–C) Pearson correlations, based on fold changes (FC) for 7,192 transcripts with an RPKM (reads per kilobase of exon per million reads mapped) >0.5. ctrl, control.
(D) qPCR for ecdysteroidogenic genes at ZT14–ZT18, control (gray) normalized to 100%. Black indicates expression in RGs isolated from PG>tim1-RNAi larvae. ∗∗p < 0.01 (Student’s t test). dib, disembodied; nev, neverland; phm, phantom; sad, shadow; spok, spookier. For primer sequences, see the Supplemental Experimental Procedures. Error bars represent 95% confidence intervals.
(E) Feeding ecdysone rescues PG>tim1-RNAi to pupal stages. EtOH, 6% ethanol alone; 20E, 20-hydroxyecdysone in 6% ethanol.
(F) Ecdysteroid titers in controls and PG>tim1-RNAi animals. Controls display a pronounced peak at 44 hr (scotophase) after the L2/L3 molt, which fails to occur when tim is knocked down. Error bars represent SD.
(G) Relationships between PG>tim1-RNAi and PG>per-RNAi RNA-seq results. Venn diagrams show overlaps between sets, based on a 3-fold cutoff, and split into up- and downregulated sets. Gene set size is given in brackets for each condition. Dotted lines indicate reciprocal relationship between day and night sets, showing the number of genes that overlap between up- and downregulated sets.
See also Tables S3 and S4.
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5. Figure 3 Timeless Interacts with PTTH and Insulin Signaling Pathways
Torso and Ras both act downstream of the neuropeptide PTTH.
(A) Overexpression of a torso-cDNA in the PG (PG>torso) causes larvae to arrest development at L1 and L2 stages and overexpression of RasV12 (PG>RasV12) results in precocious pupae that fail to eclose. Repeating these overexpression experiments in a PG>tim1-RNAi background rescues these animals to pupal (torso and RasV12) and adult stages (torso).
(B) Quantification of rescue experiments. Larval population estimates (est.) where no obvious larval lethality was observed. Error bars represent SD.
(C) Torso expression levels in controls, PG>tim-cDNA and PG>tim1-RNAi, based on qPCR. Error bars represent 95% confidence intervals.
(D) Genetic interaction between insulin pathway components and tim1-RNAi. PG> was used to drive the expression of all UAS transgenes. InRDN, dominant-negative form of insulin receptor. gskS9a encodes a constitutively active form of GSK3 (also known as Shaggy); Pi3K encodes Pi3 kinase, also known as Dp110 and Pi3K92E; akt encodes Akt1. w1118, control with same genetic background as transgenic lines.
(E) Real-time PCR of six ecdysteroidogenic genes in various genetic backgrounds. All fold changes relative to control (phm>w1118). Error bars represent 95% confidence intervals.
In (B), (C), and (E): ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < (Student’s t test).
See also Figures S3 and S4.
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6. Figure 4 IIS and the PG Clock
(A). Immunostaining of TIM in PGs isolated from controls (PG>w1118), PG>InRDN, and PG>gskS9a at ZT3 and ZT22; images were taken with the same exposure time. Scale bars, 30 μm.
(B) Quantification of fluorescence signals shown in (A). Fluorescence was analyzed with ImageJ, based on corrected total cell fluorescence (CTCF). Error bars represent SD.
(C) Model for TIM acting on PTTH pathway in response to insulin/IGF signaling (IIS) during day and night (white and gray background). Systemic insulin-like peptides (ILPs) underlie circadian control, resulting in reduced IIS during the night and elevated GSK3 activity. GSK3 stabilizes TIM, which, in turn, activates torso. InR, insulin receptor.
(D) Model for synergistic effects on larval phenotypes and ecdysteroidogenic gene expression. Photophase is shown as white; scotophase is shown as gray zones. TFs, transcription factors. WT, wild-type. Red line indicates whether an ecdysone pulse is formed or not.
See also Figures S3 and S4.
Current Biology  , DOI: ( /j.cub )
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