Norpa Signalling and the Seasonal Circadian Locomotor Phenotype in Drosophila

In this paper, we review the role of the norpA-encoded phospholipase C in light and thermal entrainment of the circadian clock in Drosophila melanogaster. We extend our discussion to the role of norpA in the thermo-sensitive splicing of the per 3′ UTR, which has significant implications for seasonal adaptations of circadian behaviour. We use the norpA mutant-generated enhancement of per splicing and the corresponding advance that it produces in the morning (M) and evening (E) locomotor component to dissect out the neurons that are contributing to this norpA phenotype using GAL4/UAS. We initially confirmed, by immunocytochemistry and in situ hybridisation in adult brains, that norpA expression is mostly concentrated in the eyes, but we were unable to unequivocally reveal norpA expression in the canonical clock cells using these methods. In larval brains, we did see some evidence for co-expression of NORPA with PDF in clock neurons. Nevertheless, downregulation of norpA in clock neurons did generate behavioural advances in adults, with the eyes playing a significant role in the norpA seasonal phenotype at high temperatures, whereas the more dorsally located CRYPTOCHROME-positive clock neurons are the likely candidates for generating the norpA behavioural effects in the cold. We further show that knockdown of the related plc21C encoded phospholipase in clock neurons does not alter per splicing nor generate any of the behavioural advances seen with norpA. Our results with downregulating norpA and plc21C implicate the rhodopsins Rh2/Rh3/Rh4 in the eyes as mediating per 3′ UTR splicing at higher temperatures and indicate that the CRY-positive LNds, also known as ‘evening’ cells are likely mediating the low-temperature seasonal effects on behaviour via altering per 3′UTR splicing.

. Results of Kruskall Wallis ANOVA and Dunn posthoc tests among the experimental (Gal4/Gal80 driver x UAS-RNAi) and control parental genotypes (driver or UAS-RNAi) crossed to w 1118 . The significant post hoc results are highlighted in blue cells (advance) or red cells (delay). When the experimental group is significantly different from both control genotypes in a consistent direction (advance or delay), both cells are the same colour.

In Situ Protocol
Initially, norpA expression has been evaluated following Wülbeck and Helfrich-Förster protocol (2007). w1118 cDNA was used as template from which the norpA probe was synthesised by a nested PCR strategy. First a large region that included the template for the norpA probe was amplified and used as a source of DNA for the subsequent PCR reactions. These were carried out utilising two sets of primers that include in their sequence either T7 or T3 RNA polymerase promoters. The product of this PCR was subjected to RNA transcription in order to obtain a sense norpA probe and an anti-sense norpA probe (Figure 3.11). In both probes, a ribonucleotide conjugated with Digoxigenin (DIG) was incorporated in the reaction in order to be recognised by a specific antibody in the following in situ steps.
Brains were rehydrated through downgrading 90%, 70%, 50%, 30% methanol/0.2% PBT series (1× PBS plus 0.2% Triton-X) and finally washed for five times in PBT. Brains were then treated with proteinase K in order to increase the probe penetration for 3 min at room temperature and subsequently for 1 h at 4 °C. Proteinase K treatment was concluded by three washes with glycine (10 mg/mL) and subjecting brains to 4% paraformaldehyde in PBS fixation. After being washed for 5 times in PBT, samples were incubated for 5 min in a 1:1 mixture of PBT:HYB (50% formamide, 5X SSC-DEPC, 0.1% Triton-X, 100 μg/mL heparin, 100 μg/mL salmon sperm DNA and 500 μg/mL of yeast t-RNA) at room temperature. This mix was replaced with HYB and brains were incubated for 2h at 60°C. Subsequently, samples were incubated with serial dilutions (50, 100 or 150 ng) of RNA probe dissolved in HYB buffer at 60 °C ON. After overnight hybridisation, the excess of probe was removed through two downgrading washes: the first of 3:2, 1:1, 2:3 ratio of HB:2XSSCT at 60 °C (10× SSC: 3 M NaCl, 0.3 M sodium citrate, DEPC H2O) and the second with a 3:2, 1:1, 2:3 ratio of 0.2× SSCT:PBT at room temperature. Subsequently brains were washed three times with TNT (0.1 M Tris HCl pH 7.5, 0.15 M NaCl and 0.05% Tween 20) before being incubated for 2 h in TNB (0.5% Blocking Buffer supplied with Tyramide Detection Kit dissolved in TNT). Brains were then incubated ON with antibody diluted in TNT (dilution 1:100). Two different set of antibody, which recognise the modified nucleotide included during the RNA probe synthesis, have been used separately: one with horseradish peroxidase epitope and another one conjugated with biotin. In the case of this latter, a secondary antibody has been used which recognises biotin and it is conjugated with horseradish peroxidise. In both cases, the luminescence signal was generated by the peroxidase which cleaves the Tyramide molecules. In order to remove the excess unbound antibody, brains were washed five times in TNT for 15 min each and, subsequently, incubated for 2-3 h in a solution containing Tyramide (Cyanine 3, diluted 1:50 in amplification buffer). At the end of the detection reaction, brains were washed three times with TNT for 15 min each and mounted in a slide using Vectashield to amplify and protect the fluorophore signal. The samples were further processed to detect PDF protein or LACZ. In this case, brains were treated with TNB buffer and the ICC protocol was followed.

Strategy Adopted to Generate the Sense and Anti-Sense Probes Used for the In Situ Hybridisation
A) full length norpA is reported including exons and introns. B and C), an enlargement of the region amplified for generating the probes is shown. Black arrows indicate the first set of primers utilised for amplifying a larger region that has been used as template for the following PCR reactions using the T7 and T3 probes (different coloured arrows).

Immunocytochemistry in Drosophila Brain (ICC)
Dissected brains were washed for 5 min in 75% MetOH in PBT (PBS plus 0.5% of Triton-X). Two subsequent 5 min washes were performed with 50% and 25% MetOH in PBT, respectively, and finally three 5 min washes with 0.5% PBT in order to be permeabilised for the following steps. Subsequently, dissected brains were blocked for 2 h with 1% Bovine Serum Albumin (BSA) to minimise non-specific binding of the primary antibody. After blocking, the brains were incubated with the primary antibody which was diluted in 1% PBT which also contained 1% BSA and 0.1% Sodium azide. The presence of sodium azide prevents the contamination of the antibody solution with bacteria and thereby allows the re-use of the antibody. The brains were incubated in primary antibody for the required period of time (between 1 and 3 days) at 4 °C every day mixing by pipetting the solution. They were then washed five times for 5 min each in 1% PBT. After being washed the brains were incubated for 2 h at room temperature in the appropriate secondary antibody (raised again the animal in which the primary antibody was generated) which was conjugated with a fluorophore. Once the protein of interest had been labelled with the appropriate antibody, the brain was mounted onto slides (VWR) with a drop of mounting medium which comprises of 80% glycerol and 3% propyl gallate and covered with a coverslip of 0.1 mm thickness (VWR). The slides were stored in the dark at 4 °C. They were visualised on an Olympus FV1000 confocal microscope. Individual images were taken of planes at different depths in order to create a Z-series for each brain. The size of the sections forming a Z-series was either 0.44 μM (if using 40× objective) or 1.4 μM (if using 20× objective). The optimal microscopic settings, in particular the laser gain, amplifier gain and offset and laser intensity were adjusted for each experiment in order to maximise the quality of the images. Dissected larval brains were treated similarly except that 3 rd instar larvae maintained at 18 °C.

Primers Used for RT-PCR of plc21C
Primer DNA sequence and numbering Reference plc21C Figure S1. NORPA and PER-reporter expression. Representative R32 Drosophila brain (20×, A) and enlargement of the right hemisphere (B). Confocal images were merged together in order to create a full tri-dimensional structure of the brain. The red signal (Cy5) corresponds to lacZ (PER reporter) whereas green (Cy2) identifies NORPA expression.
A B Figure S2. NORPA is expressed in the optic lobes. (A). w 1118 brain labelled with PDF (red; Cy5) and NORPA (green; Cy2) antibodies. The brain presented is an image of 30 independent layers merged together. (B). norpAp41; R32 adult brain. Confocal images were merged together in order to create a full tridimensional structure of the brain. The red signal (Cy5, in the top left panel) corresponds to LACZ antibody whereas green signal (Cy2, right top panel) represents NORPA antibody.     Figure S8. Effects on locomotor activity of knockdown of plc21C. Mean (+/-sem) locomotor activity events per 30 min time bin are plotted against ZT in an LD12:12 schedule, ZT0-12 day, ZT12-24 dark. Blue trace is the experimental genotype (Gal4 x plcCi), brown is w x UASplcCi, and green is w x Gal4. Kruskall Wallis ANOVA followed by Dunn a posteriori procedure was applied to EZt50 and MZt50 values. There were no examples where the experimental (blue) genotype was significantly different from both controls.