Interaction of obesity linked AP-2 and CG10440 in the regulation of the Octopaminergic system
Search for function using Drosophila melanogaster’s genetics
JAYASIMMAN RAJENDRAN
Degree Project E in Applied Biotechnology 45 hp, 2012
Department of Neuroscience, Functional Pharmacology, BMC, Uppsala University, Uppsala, Sweden.
Supervisor: Prof. Dr. Helgi Schiöth
Co-supervisor: Dr. Michael J Williams, Senior Research Fellow.
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Abstract
Genetic obesity is caused due to the interaction of environmental factors with multiple gene functions. The genes, transcription factor activator enhancer protein 2 beta (TFAP2b) and potassium ion channel tertramerization domain containing protein 15 (KCTD15) have been linked with obesity but the functions of these genes in the regulation of obesity are unknown.
Orthologous of these genes can be found in Drosophila melanogaster (AP-2 and CG10440 respectively) and in the current study their role in the regulation of the octopaminergic system has been investigated by epistatic analysis. Flies with reduced AP-2 and CG10440 expression in octopaminergic (Tdc2) neurons displayed more male-male courtship behavior. Over- expression of AP-2 led to an increase in aggression and foraging behavior, as well as a decrease in mating behavior, lipid storage and starvation resistance. However, in the absence of CG10440, the AP-2 over-expression phenotype returned to normal levels. This indicates that CG10440 is required for the functional activation of AP-2 for the proper regulation of the octopaminergic system.
Key words: AP-2, CG10440, Octopamine, Drosophila melanogaster, Aggressive behavior.
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Acknowledgement
First of all, I wish to express my honorable gratitude to my supervisor Prof. Helgi Schiöth, Head of the functional pharmacology in Department of Neuroscience, Uppsala university, for giving me the opportunity to work in his group to acquire more knowledge and experience in research, he motivated me towards my goal and success.
I would like to thank my co-supervisor Dr. Michael J Williams, Senior research fellow, for his valuable guidance and support. He inspired me very much to work in this project. In addition, I would like to thank Mr. Philip Goergen, for his willingness to assist me with valuable ideas and suggestions for experimental setup in my project, and also I thank to all my fellow mates in the Laboratory for their timely assistance.
Finally, I would like to thank my course coordinator Prof. Staffan Svärd and Dr. Britta Stadelmann, Department of Cell & Molecular Biology, and all others who supported me to complete this project successfully.
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Abstract...
Acknowledgement...
1. Introduction…...
1.1. Drosophila melanogaster life cycle.…...
1.2. Obesity linked genes: TFAP2 and KCTD15...
1.3. Drosophila Transcription factor AP-2...
1.4. Drosophila gene CG10440……...
1.5. Interaction of AP2 and KCTD family proteins…...
1.6. Regulation of AP-2 in the octopamine pathway...
1.7. The effect of octopamine on fly behavior...
1.8. Aim ………
2. Materials & methods
2.1. Fly stock...
2.2. Crosses...
2.3. Aggression assay...
2.4. Mating behavior assay...
2.5. Capillary Feeding (CAFE) assay...
2.6. Feeding after starvation...
2.7. Starvation resistance assay...
2.8. Triglyceride assay for Lipid storage...
2.9. RNA Extraction...
2.10. cDNA synthesis...
2.11. Real time PCR...
2.12. Statistical analysis...
Pg.no 2 3 6 7 8 8 9 10 10 11 12 13 13 13 13 14 14 15 16 16 17 17 17 18
5 3. Results
3.1. The effect of AP-2 and CG10440 on activity and aggressive behavior…..
3.2. The effect of AP-2 and CG10440 on male-female courtship behavior…..
3.3. The effect of AP-2 and CG10440 on feeding behavior...
3.4. The effect of AP-2 and CG10440 on feeding after starvation...
3.5. The effect of AP-2 and CG10440 on starvation resistance...
3.6. The effect of AP-2 and on lipid storage...
3.7. Relative gene expression studies on AP-2, CG10440 and TBH by real time PCR...
4. Discusson …...
5. Annexture
Annexure 1: Protocol for real time PCR reaction…...
Annexture.2: Primer sequences and working annealing temperature...
Annexture.3: genetic map to generate AP-2 over expressed in CG10440 knocked down flies………....
References...
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1. Introduction
Obesity is a very common disease affecting human beings. Today, 63% of the world’s population is affected by overweight and obesity. This is caused by high calorie intake and reduced physical activity. High BMI or obesity is associated with insulin reduction and free fatty acid deposition in the blood, and these lead to chronic cardio-vascular diseases with increased blood pressure, lipid storage, diabetes and other health problems.
Genetic obesity problems are caused by the interaction or influence of environmental factors on the functions of multiple genes, affecting metabolic pathways, which lead to overweight. There are a number of genes that are involved in the regulation of several neuronal signal transduction and signal coordination pathways within the central nervous system (CNS) to regulate metabolism for proper energy expenditure (Gabriele et al., 2006).
Abnormal expression or improper functioning of these genes in the neuronal system can affect the metabolism.
There have been many obesity linked genes identified so far which seem to show small generic variance in the development of obesity. Gene loci located near FTO, MC4R, TMEM18, GNPDA2, SH2B1, KCTD15, MTCH2, NEGR1, BDNF, ETV5 and TFAP2B are associated with obesity and are also involved in the distribution of lipid storage and fat body (Lindgren et al., 2009; Willer et al., 2009). A lot of research has been carried out to understand the function of these genes in obesity using different study models. Humans are the most studied species, followed by laboratory research animals like rat, mice and flies.
About 75% of the genes in humans that are disease prone have been found to be homologue in Drosophila (Lloyd and Taylor, 2010). Also, adult flies have physical parts whose functions are similar to the human heart, lung, kidneys, gut, reproductive tract and brain. Moreover, complex behaviors like sleep, learning, memory, feeding, aggression, mating, flying and circadian rhythms are regulated by more than 100,000 neurons in the Drosophila neuronal system. The central nervous system of the fly responds in a similar fashion to the mammalian system when it is subjected to various drugs (McClung and Hirsh, 1998; Moore et al., 1998;
Bainton et al., 2000; Nichols et al., 2002; Rothenfluh and Heberlein, 2002; Satta et al., 2003;
Wolf and Heberlein, 2003; Andretic et al., 2008). All of these studies strengthen the fact that the fly can be used as a model to study human disease, and functions of orthologous genes.
7 1.1. Drosophila melanogasters Life cycle
Drosophila melanogaster is a fruit fly which is ~3 mm in size. Its life cycle is short (2 weeks) and the embryos are developed into larva one day after fertilization. After that these develop into 3rd instar larva within 2 days (Fig.1a). It remains in the same stage for another 2 days and then form the pupa (Fig.1b), the complete adult fly is formed in 4 days from the pupa stage.
The wild type fly has yellowish brown body with oval wings and red eyes. The female flies are larger than the male flies and they can be differentiated by black markings in their abdomens (Fig.1c&d). Normally flies are raised in 10 days at 25°C. However, their generation time can be increased by decreasing the temperature thereby slowing down the growth. The minimum temperature suitable for growth is 18°C. They can grow up to 12-13 days at 21-22°C, and 19 days at 18°C. The newly enclosed virgin females will remain virgin for 6 hours at 25°C, 12 hours at 21-22°C and 18 hours at 18°C. These virgins can be differentiated from adult female by locating the meconium (dark mark) in the front side of the abdomen (Fig.1e).
1.a) 1. b) 1. c)
1.d) 1. e)
Figure.1: Drosophila malanogaster: a) Instar larvas, b) pupe, c) male adult fly with dark black mark on abdomen, d) female adult fly with dark bands on abdomen and e) male and female virgins with meconium (circled) in front side of the abdomen.
The understanding of the Drosophila genetics is not complicated; it has only 4 pairs of chromosomes. The first chromosome is sex chromosome X/Y, the others are autosomes and are numbered as 2, 3 and 4. Each autosome has two arms termed as Left (L)
8
and Right (R). These chromosomes are mapped with 102 numbered bands which are used to identify the location of genes. The sex chromosome is mapped with first 20 bands, 2nd chromosome with 21-40 in left arm and 41-60 bands in right arm, 3rd chromosome is mapped with 61-80 bands in left and 81-100 bands in right arms and 4th chromosome is tiny and mapped with rest of 2 bands. Each numbered band is further divided into six lettered bands A- F. Each lettered band is subdivided into 13 numbered divisions which have 300bp DNA and 15-25 genes.
The entire Drosophila genome was sequenced in the year 2000 and mutant flies with defects in several of their genes are available for research purposes today. Many conserved genes are similar to Human genes, both share similarities in basic cellular structure and function, for example they share similar functions in intracellular signaling pathways (Pawson and Bernstein, 1990), patterning (Krumlauf, 1992), behavior (Kandel and Abel, 1995), tumor formation and metastasis (Potter et al., 2000), development and degradation of Neural system (Fortini and bonini, 2000) and behavioral effect of drugs and neurotransmitters (Bianton et al., 2000, Li et al., 2000). So the function of any Drosophila gene which is shared with humans can be studied by using Drosophila genetics.
1.2. Obesity linked genes TFAP2B and KCTD15
The human genes TFAP2B and KCTD15 are associated with obesity and relationship between metabolic syndromes. It has been reported that the loci near the TFAP2B is strongly associated with adiposity and fat distribution (Lindgren et al., 2009). The Genome wide associated study (GWAS) have identified several Single Nucleotide Polymorphisms (SNPs) which influence the body mass index and that have been reported to be located at or near genes expressed in the brain (KCTD15) or involved in cell apoptosis and proliferation (TFAP2B) (Mei et al., 2012; Hong et al., 2011). These pose a strong evidence to the fact these genes are associated with obesity.
1.3. Drosophila Transcription factor AP2
The Drosophila gene CG7807, termed as AP-2 protein is orthologous to the human Transcription factor Activator enhancer protein family member TFAP2B. Members of the TFAP2 family are sequence specific DNA binding proteins involved in the regulation of transcription, either in transcriptional repression or activation. In humans, five protein members have been identified so far, they are TFAP2A, TFAP2B, TFAP2C, TFAP2D and
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TFAP2E. They each have a unique structure and function (Moser et al., 1995; Chazaud et al., 1996; Oulad-Abdelghani et al., 1996; Zhao et al., 2001; Wang et al., 2004; Feng and Williams, 2003). They have helix-span-helix dimerization motifs at the carboxyl terminus that binds with GC rich sequence 5’-GCCNNNGGC-3’ during transcription. Transcription is activated by proline-glutamine rich domain in the amine terminus. The basic region is located in between these domains. (William and Tjian, 1991a and 1991b).
Generally AP-2 proteins are involved in the development and morphogenesis of tissues and organs. In this family AP-2α, AP-2β and AP-2γ are located in the nervous system.
AP-2α is involved in the development of Noradrenergic (NA) neurons, so they fail to develop properly in the mutants lacking AP-2α (Holzschuh et al., 2003). AP-2β is involved in the differentiation of Neural crest stem cells (NCSC) to a sympathoadrenal (SA) cell fate, and also it is involved in the synthesis of Noradrenaline and NA-synthesizing Dopamine β- hydroxylase. Ap-2γ does not affect any generation of melanocyte or SA cells (Seok Jong Hong et al., 2008). In the Drosophila embryo, AP-2 is expressed in maxillary segment, neural structures and the central nervous system (CNS). During larval stages it is expressed in the legs, antennal and labial imaginal disks (Bauer et al., 1998; Monge and Mitchell, 1998). A mutation in AP-2 causes defects during probiosis development and leg joint formation (Monge et al., 2001; Kerber et al., 2001). In mammals, AP-2β affects CNS crest and limb mesenchyme which disturb the facial and limb development (Schorle et al., 1996; Zhang et al., 1996).
1.4. Drosophila gene CG10440
The mRNA sequence of Drosophila gene CG10440 is predicted to have a Broad complex, Tramtrack and Bric-a-brac (BTB) domain which is similar to Potassium channel tetramerization domain (KCTD) protein family members. In humans, KCTD family has 21 members; they are involved in several important biological processes. The gene CG10440 in Drosophila is orthologous to human KCTD15 and KCTD1, which are expressed in the gastrointestinal tract, gingival in oral cavity, skin, uterus in female reproductive system, kidney, embryo and fetal heart. In zebrafish, Kctd15 was shown to be necessary for neural crest cell development. The expression of Kctd15 in embryos has been observed in zebrafish and xenopus and has also been identified in humans (Hotta et al., 2009; Willer et al., 2009). .
10 1.5. Interaction of AP-2 and KCTD family proteins
There is some evidence showing that the AP2 family of proteins interacts with KCTD family members, The Interaction of KCTD1 with AP-2α was observed in a yeast two-hybrid system (Giot et al., 2003). Furthermore, AP-2α mediated trans-activation was repressed through interaction with the KCTD1 BTB domain and it was considered as a negative regulator of AP- 2α (Xiaofeng et al., 2009). Similarly, the promoter activity and expression of KCTD10 was stimulated by Transcription factor specificity protein 1 (SP1) binding, but the AP-2α binding to the promoter region showed the opposite effect on KCTD10 expression (Rushi et al., 2009). These results suggest that there are the possible interactions between AP2 and KCTD- family members. AP-2 and CG10440 genes in the Drosophila melanogaster are members of TFAP2 and KCTD family respectively, so there is a possibility of interaction between AP-2 and CG10440 to regulate bio-molecules in various metabolic pathways.
1.6. Regulation of AP-2 in the octopamine pathway
The AP-2 protein may be involved in neurotransmitter synthesis pathways such as octopamine and dopamine in the nervous system. The AP-2 binding site is located in the upstream promoter region of Tyrosine Hydroxylase (TH) gene and domain III of Dopamine Beta Hydroxylase (DBH) promoter and also includes transcription start site and GC rich sequences. Thus it regulates the transcriptional activity of TH and DBH in noradrenergic or adrenergic neurons in both central and pheripheral catecholamenergic neurons (Hee-sun Kim et al., 2001). It has been indicated that AP-2 is involved in dopamine synthesis. In octopamine synthesis, Increase in tyrosine decarboxylase (Tdc) or tyramine beta hydroxylase (Tbh) enzyme level in the neural system triggers elevated secretion of octopamine (Fig.2). If AP2 is involved in the octopamine pathway, modulated expression of AP2 may influence octopamine synthesis. The activity of AP-2 proteins can be suppressed or activated by many ways such as altering trans-activation potentials, DNA binding regions, subcellular localization (Aqeilan et al., 2004; Mazina et al., 2001; Pellikainen et al., 2004) and degradation (Li et al., 2005;
Nyormoi et al., 2001). The mechanism of regulation can be controlled at the molecular level by post-translational modifications, including protein kinase A mediated phosphorylation (Garcia et al., 1999; Park and Kim, 1993), redox regulation, sumoylation (Zhong et al., 2003) and physical interaction with proteins.
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Figure.2: Dopamine and Octopamine pathway in the neuronal system. Dopamin and octopamine both are synthesized from the same amino acid tyrosine. In the dopamine pathway, tyrosine is converted to dopa by tyrosine hydroxylase (TH) then carboxylated to dopamine. Finally it is converted nor-ephenepherin. In octopamine synthesis tyrosine is decarboxylated to tyramine by tyrosine decarboxylase and converted to octopamine by tyramine decarboxylase. Increase in tyrosine decarboxylase (Tdc) or tyramine beta hydroxylase (Tbh) enzyme level in the neural system triggers elevated secretion of octopamine.
1.7. The effect of octopamine on fly behavior
Octopamine is a type of monoamine present in insects and has a function similar to mammalian nor-adrenaline. Octopamine is synthesized from the amino acid tyrosine.
Tyrosine is decorbaxylated to tyramine by the enzyme tyrosine decarboxylase (Tdc) and followed by beta-hydroxylation to octopamine by tyramine beta hydroxylase (TBH).
Octopamine acts as a neurotransmitter regulating physiological processes like sensory signaling, ovulation, egg laying, fight or flight response, memory and learning (Roeder, 2005). Octopamine is required in adult flies to stimulate aggressive behavior (Hoyer et al., 2008). Other than Drosophila melanogaster, octopamine influences the aggression in crickets (Stevenson et al., 2005) and foraging behavior in honey bees (Barron et al., 2002). Thus octopamine influences the behavior of the fly by regulating signaling in receptor expressed cells.
Tyrosine
Tyramine
Octopamine
Dopa
Dopamine
Nor-ephenephrine
Tyrosine hydroxylase
Dopa carboxylase
Dopamine
beta hydroxylase Tyrosine decarboxylase
Tyramine beta hydroxylase
12 1.8. Aim:
Epistatic analysis was carried out with intention to determine the function of AP-2 and CG10440 in octopaminergic system. These gene expressions are modulated in Tyrosine decarboxylase 2 (Tdc2) expressed regions of fly brain by using RNA interference and UAS-Gal4 system (Joseph B Duffy., 2002). Tdc2 is expressed in the antennal lobe and subesophegeal gangilion of the fly brain (Christopher et al., 2008), so mutations of genes in these regions affect fly behaviors such as aggression, fly activity, courtship, foraging, starvation resistance and lipid storage. The function of these genes in octopamine synthesis is predicted by analyzing behavioral phenotype.
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2. Materials and methods
2.1. Fly stock
The expressions of both AP-2 and CG10440 are modulated in octopaminergic neuron by using Tdc2-Gal4 driver flies, Tdc2 (Tyrosine decarboxylase2) is expressed in the octopaminergic neuron in the central nervous system of fly brain. The GAL4 driver fly Tdc2- Gal4, wild type yw, UAS-CG10440ir, and AP2 over-expressed fly UAS-AP2OE were ordered from the Bloomington stock centre Indiana USA, and the AP2 inverted repeats for knockdown UAS-CG7807 ir flies were ordered from VDRC, Vienna, Austria.. They were cultivated on Jazzmix standard fly food containing brown sugar, corn meal, 10% yeast, agar, benzoic acid, methyl paraben and propionic acid and maintained at 25ºC in an incubator on a 12:12 light:dark cycle.
2.2. Crosses
Tdc2-Gal4 driver flies were crossed with yw to generate a recombinant fly (“yw;Tdc2-Gal4”) and it was served as a control for the whole experiment. Tdc2-Gal4 driver flies were crossed with UAS-CG7807ir, UAS-CG10440ir and UAS-AP2OE to knocked-down AP-2, CG10040 and over-expressed AP-2 respectively, then the AP-2 over-expressed in CG10440 knock-down flies were produced with help of double balancer using generation crosses (Annex.3). The experimental flies were grown at 29ºC from the larval stage in incubator on a 12:12 light: dark cycle.
2.3. Aggression assay
The dimensions of the cylindrical behavioral chamber are 2 cm by 2.5 cm (height x diameter).
They were filled with 1% agarose up to 1.5 cm in height to maintain proper humidity. Newly eclosed experimental and control virgin male flies were collected and isolated on satiated condition for 5 to 7 days. The behavioral tests were carried out at room temperature with appropriate humidity. Two isolated male flies were anesthetized using CO2 then transferred into a behavioral chamber. A camera (Panasonic HDC-SD90) was positioned above the chamber and flies activity was videotaped for 20 min.
The behaviors was measured and patterns were categorized as High intensity fighting (wing threat (Fig.3a), boxing, lunging and chasing), Low intensity fighting (Wing flick and pushing (Fig.3b)) and courtship behavior (one wing out (Fig.3c), circling, tapping from behind (Fig.3d), licking back (Fig.3e), and Abdomen bend (Fig.3f)) (Chen et al., 2002). The
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activity of flies was measured by calculating total time of fly movement in 20 min and ten replicates were conducted.
3.a) 3.b) 3. c)
3.d) 3. e) 3. f)
Figure.3: Aggression and courtship behaviors of Drosophila melanogaster. a) High intensity fighting behavior-wing threat, b) Low intensity fighting behavior – pushing, c) courtship behavior – one wing out or singing, d) courtship behavior – tapping from behind, e) courtship behavior-licking back and f) courtship behavior- abdomen bend is copulation behavior.
2.4. Mating behavior assay
Newly eclosed male virgins were collected and isolated in individual food vials and aged for 5 to 7 days. The male was then put in the behavioral chamber with a 2-days-old virgin yw female fly. The behavioral activity was videotaped for 10 min. Latency, Courtship index, as well as the frequency of mating behaviors were measured. Latency was calculated by counting time taken by fly to initiate mating, and Courtship index was calculated by finding time duration for male fly mating with female. Ten replicates were conducted.
2.5. Capillary feeding (CAFE) assay
This method was modified from Willam et al. 2007. A transparent plastic cylindrical vial 9 cm by 2 cm (height X diameter) was taken containing 1% agarose (5cm high) to provide moisture and humidity for the flies. A calibrated capillary glass tube (5μl, catalog no. 552- 0043; VWR) was filled with liquid food which contained 5% of sucrose, 5% yeast extract, 0.5% food coloring dye. A layer of mineral oil was used to prevent the liquid food
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evaporation from the capillary tube (Fig.4a). The five males, 5-7 days older flies, were put inside the chamber and the opening of the vial was covered with parafilm and a capillary tube was inserted from the top through parafilm. The experimental set up was kept at room temperature and fly activity was videotaped for 24 hours using a HD camera (Panasonic SDS90). The initial and final food level in the capillary tube was marked to observe total food intake per day. The number of feeding bouts per fly were observed from the recorded video (Fig.4b), and the average meal size of the fly was calculated from ten replicates.
4.a) 4.b)
Figure.4: Foraging and feeding behavior by CAFE assay. a) Capillary tube filled with immersive oil and liquid food. The difference in food height between second and third capillary tube shows the food taken by the flies. B) Figure shows the fly feeding by capillary in CAFE assay method.
2.6. Feeding after starvation
This method was modified from Al-Anzi et al. 2010 to study feeding behavior of flies.
Twenty male flies, 5-7 days-old, were kept in a vial containing 1% agarose and starved for 18 hours. Then they were transferred to normal a food vials (5% sucrose, 5% yeast extract and 1% agarose) and were allowed to feed for 15 min. Afterwards the flies were transferred to a second food vial which contained 2% blue food coloring dye with normal food (5% sucrose, 5% yeast extract and 1% agarose). The flies’ abdomens were observed under the dissecting microscope and the colored crop was scored. The percentage of over-fed flies were calculated by counting the blue colored crop of the flies (Fig.5). Five replicates were conducted.
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Figure.5: Foraging behavior after starvation. The colored abdomen flies after fed with blue colored dyed food.
2.7. Starvation resistance assay
The starvation resistance or percentage survival during starvation was measured by this method. Twenty male flies, 5 to 7 days old were transferred in to a vial containing 5 ml of 1%
agarose which provides water and humidity but no food source for the fly. The vial was kept at 25ºC in an incubator on a 12h:12h light:dark cycle. The numbers of dead flies were counted every 12 hours. This allowed calculating the median time of death and a graph was plotted with % starvation resistances against time. Five replicates were conducted for each strain.
2.8. Triglyceride assay for Lipid storage
The flies (25 males) were homogenized with 100 µl of PBST buffer (1x phosphate buffer with 10% Tween20), incubated at 70ºC for 5 min and then centrifuged at maximum speed for 10 min. The supernatant was transferred into a new eppendoff and used as samples. The glycerol standard was used to generate a standard curve with concentration of 1.0, 0.8, 0.6, 0.4, and 0.2 mg/ml equivalent triolein concentration. 100 µl of free glycerol reagent was added with 10µl of blank (PBST), standards or samples and initial absorbance at 540nm was measured after incubation at 37°C for 15 min. The concentration of free glycerol in the samples was calculated from a standard curve generated by this initial absorbance values. Then, 20 µl of triglyceride reagent was added in each standard and samples and incubated at 37°C for 15 min. The final absorbance was taken at 540 nm to calculate triglyceride concentration from generated standard curve. The protein concentration of each sample was measured by the Bio- Rad protein assay kit. The concentrations of free glycerides and Tri-glycerides in samples (mg per mg of protein) were calculated from five replicates.
17 2.9. RNA Extraction
Phenol-Chloroform extraction method was used for RNA extraction from fly heads (Chomczynski & Sacchi 1987). Fifty fly heads were homogenized with 800 µl TRIzol (Invitrogen, USA), 200 µl Chloroform (Sigma Aldrich) was added and samples were centrifuged at 12000 rpm for 15 min at 4°C. The RNA containing aqueous layer was separated with 500 µl isopropanol (Solvaco AB, Sweden) at -32°C for 12 h to precipitate the RNA. Samples were then centrifuged at 12000 rpm for 10 min at 4°C to collect the RNA pellets, and washed with 75% ethanol (Solvaco AB, Sweden) to remove the organic impurities, it was air dried to remove the traces of ethanol. The RNA pellets were then dissolved in 30 µl RNAse free water (Qiagen GmBH, Germany), and were incubated at 75°C for 15 min to dissolve RNA. 2 µl DNAse I (10 U/µl) (Roche GmBH, Germany) was added to the samples to remove DNA contamination by incubating at 37°C for 3 h. The enzyme was deactivated by incubating at 75°C for 15 min. Removal of DNA was confirmed by PCR using Taq polymerase 5U/µl (Biotools B & M Labs, Spain) followed by gel electrophoresis (2%).
The RNA concentration was measured using nanodrop ND 1000 spectrophotometer (Saveen Werner).
2.10. cDNA synthesis
cDNA was synthesized from RNA template using dNTP 20 mM (Fermentas Life Science), random hexamers as primers and M-MLV Reverse Transcriptase (200 U/µl) (Invitrogen, USA) by incubating in a PCR machine at 25°C for 10 min for the annealing of random hexamers, then incubated at 37°C for 1 hour for elongation and followed by 95°C for 15 min to inactivate enzymes.
2.11. Real time PCR
Real time PCR was performed for all the genes of interest (AP-2, CG10440 & TBH) and three housekeeping genes (RPL32, EF1 & TUB) with SYBR-Green reaction mix (Invitrogen, USA). The RT PCR was run with 5 µl cDNA template (5 ng/ml), 0.05 µl of each forward and reverse primer, 2 µl of 10X buffer, and 0.08 µl of Taq Polymerase per sample. Every reaction was run for 55 cycles with 20 second denaturation at 95ºC, 30 sec annealing at specific primer annealing temperature (varies with different primer depending on its length and CG concentration) and 40 second elongation at 72ºC. Melting curve analysis was done with start temperature at 55ºC and end temperature till 95ºC (Annex.1).
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The Relative Fluorescent obtained from the real time data was used to calculate the cycle threshold (Ct) values for each sample. The individual primer efficiencies were calculated using LinReg PCR tool and they were analyzed with Grubb’s test to detect and remove any possible outlier and calculate the mean and standard deviation of primer- efficiency. The primer-efficiency was used to calculate corrected Ct or ‘actual fold difference using a positive control as calibrator. The concentration of each sample was calculated using a positive control of known concentration (usually 5ng/µl). Then the normalization factor for individual sample was calculated by running real time PCR for three housekeeping genes RPL32, EF1 and TUB (Annex.2). The minimum Ct value was used as calibrator to calculate the corrected Ct values. Corrected Ct values of all samples from all three housekeeping genes were used to calculate the Normalization factor using GeNorm application (v3.3, Centre for Medical Genetics). Normalization factors for individual sample were used to calculate the normalized Ct for genes AP-2, CG10440 and TBH. The normalized Ct values were then used for statistical analysis to check gene expressions.
2.12. Statistical analysis
The mean and standard deviation from all replicates of each experiment were calculated. The data were analyzed and differences between groups were identified by one way ANOVA test followed by Bonferroni comparison of all pairs post test. All the statistical analysis was carried out with GraphPad Prism software.
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3. Results:
3.1. The effect of AP-2 and CG10440 on activity and aggressive behavior:
Activity and aggression are very important for animals to survive, and thus these behaviors were measured in flies whose AP-2 and CG10440 gene expressions were modified. The activity was not affected when AP-2 and CG10440 were down regulated in octopamine neurons, however over expression of AP-2 in the same neurons showed significantly higher activity (Fig.6a). Aggressive behavior of the fly was determined by analyzing and measuring activities like high intensive fight, low intensive fight and male-male courtship behavior.
Over-expressed AP2 flies showed increased high intensive fighting behavior (P<0.0001) when compared to others, where fencing (P<0.0001) and lunging (P<0.001) were higher (Fig.6b.i&b.ii). During low intensity fights, only the flies with CG10440 knock-downs showed a higher number of wing flicks, but overall percentage of low intensity fights was not affected (Fig.6c.i&c.ii). AP-2 knock-downs showed significantly higher male-male courtship behavior (P<0.05) when compared to AP-2 over-expression. These data suggest that the activity and aggression were increased when AP-2 was over-expressed in the octopaminergic neurons. However, when AP-2 was over-expressed in the absence of CG10440 in octopaminergic neurons, the fly’s activity and aggression were different from over-expressed AP-2 phenotype having normal amount of CG10440. It's activity was significantly lower (P<0.05); high and low intensive fights did not differ and male-male courtship behavior was increased (P<0.05) when compared to the flies with AP-2 over-expression.
Over-expression of AP-2 in the absence of CG10440 in octopaminergic neuron did not induce any aggression, and this indicates that CG10440 is probably required for the functioning of AP-2 in octopaminergic neuron.
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6.a) 6.b.i)
Control dAP2 -
CG10440 - dAP2 +
dAP2 +; CG10440 - 0
20 40 60 80 100
________________
| __________|
| _____|_____
| | |
***
***
*** *
% Activity
Control dAP2 -
CG10440 - dAP2 +
dAP2 +; CG10440 - 0
20 40 60
***
% Total high intensive fight
6.b.ii)
Wing threat
Fencing
Lunging
Chasing 0
10 20 30
control dAP2- CG10440- dAP2+
dAP2 +; CG10440 -
***
**
High intensive behaviors
% behavior
6.c.i) 6.c.ii)
Control dA
P2 - CG1
0440 - dA
P2 +
dAP2 +; CG1 0440 - 0
20 40 60 80
% Total low intensive fight
Wing
flick
Pushing 0
20 40
60 control
dAP2- CG10440- dAP2+
dAP2 +; CG10440 - _________
| ____ |
|__ |
| | ___
| | |* * **
*
*
** ***
______
| ____|
| | | __
| |
**
*
% low intensive fight behavior
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6.d.i) 6.d.ii)
Control dA
P2 - CG1
0440 - dA
P2 +
dA P2 +; CG1
0440 - 0
20 40
60 __________ _____
| | |
* *
% Total male-male courtship behavior
On e win
g out
Circling
Abdome n bend
Tapping from behind
Licking 0
10 20 30 40
control dAP2- CG10440- dAP2+
dAP2 +; CG10440 - ______
| ___|
| | __ _|
_ |_ | |
| | |
***
***
*** ***
*** **
% Male-male courtship behavior
Figure.6: Effect of AP-2 and CG10440 on activity and aggressive behavior. The aggression assay was conducted between 5 to 7 days old isolated male flies, different fly behaviors were observed and percentage behaviors were plotted against strains with mean and SEM. a) percentage activity of flies, b.i) percentage high intensive fight b.ii) different behaviors of high intensity fight, c.i) total percentage of low intensive fight, c.ii) different low intensity fight behaviors of male fly, d.i) total percentage of male-male courtship behavior and d.ii) individual male –male courtship behavior influenced in fly. dAP2-: AP-2 knockdown in Tdc2 neuron, CG10040- : CG10440 knockdown in Tdc2 neuron, dAP-2+: AP-2 over-expressed in Tdc2 neuron and dAP-2+;CG10440-:
over expressed AP-2 in the absence of CG10440 in Tdc2 neuron. Significant difference between the groups:
P<0.05(*); P<0.001(**) and P<0.0001(***).
22
3.2. The effect of AP-2 and CG10440 on male-female courtship behavior:
Mating of the male with the female is a per-requisite for reproduction. The mating behavior of the fly was measured by behaviors involved during mating. The eagerness of the male was measured by looking at latency and calculating the courtship index. The AP-2 and CG10440 knock-down flies showed a significantly higher number of mating behaviors when compared to the control (P<0.05), but over-expression of AP-2 reduced the mating behavior strongly (Fig.7a.i&7a.ii). AP-2 knock-down flies did not affect the latency but showed more courtship index than controls (P<0.05), CG10440 knock-down did not affect both latency and courtship index, but over-expression of AP-2 increased the latency period (P<0.001) and reduced courtship index. The data suggests that both AP-2 and CG10440 knock-downs showed similar phenotype in mating behavior and flies with over-expressed AP-2 showed less interest in mating than the controls, AP-2 and CG10440 knock-downs. The over-expression of AP-2 in the absence of CG10440 showed an equal number of mating behavior similar to the control, but a significantly lower latency (P<0.05) and higher courtship index (P<0.05) than AP-2 over-expressed flies.
7.a.i)
On e win
g out
Circling
Abdome n bend
Tapping from behind
Licking 0
20 40 60 80 100
Control dAP2 - CG10440 - dAP2 +
dAP2+;CG10440-
***
% Mating behaviors
23 7.a.ii)
Control dAP2 -
CG10440 - dAP2 +
dA
P2 +; CG10440 - 0
20 40 60 80
__________
|_____ |
| | *
*
____
____ |_____|
| | ***
***
Total number of mating behaviors
b) c)
Control dAP2 -
CG10440 - dAP2 +
dAP2 +; CG10440 - 0
200 400 600
_________________
| ___________|
| _____ |_____
| | |
**
**
** *
Mating latency (Sec)
Control dAP2 -
CG10440 - dAP2 +
dAP2 +; CG10440 - 0
20 40 60 80 100
__________
| _____|_____
| | |
* ***
*** * _____
| |
% Courtship index
Figure.7: Effect of AP-2 and CG10440 on male-female courtship behavior. The Mating assay was conducted between 5 to 7 days old isolated male with virgin female fly. Different mating behaviors were observed and percentage behaviors were plotted against strains with mean and SEM. dAP2-: AP-2 knockdown in Tdc2 neuron, CG10040- : CG10440 knockdown in Tdc2 neuron, dAP-2+: AP-2 over-expressed in Tdc2 neuron and dAP- 2+;CG10440-: over expressed AP-2 in the absence of CG10440 in Tdc2 neuron. a.i) different behaviors involved during mating, a.ii) Number of mating behavior, b) latency and c) courtship Index. Significant difference between the groups: P<0.05(*); P<0.001(**) and P<0.0001(***).
24
3.3. The effect of AP-2 and CG10440 on feeding behavior:
Food procurement is a necessity for all living beings to acquire nutrients for their metabolic activity and body functions. The Capillary feeding (CAFE) assay was conducted in all experimental and control flies. The feeding pattern was observed by calculating number of feeding bouts per day (feeding frequency) and meal size per intake. The number of feeding bouts was counted for each fly from the 24 hours recorded video of CAFE assay. Significant difference in feeding frequency were observed in AP-2 over-expressed flies, Flies with AP-2 over expressed went for more feeding bouts than control and both knock-downs (p<0.001) (Fig.8a) (Table 3). The meal size per intake was calculated from the total food intake and feeding frequency. It is indicated that AP-2 knock-down flies fed approximately with double the volume of meal per intake (P<0.05) when compared to AP-2 over-expression (Fig.8b).
The result of CAFE assay suggests that knock down of AP-2 in octopaminergic neurons increases feed meal size, but when it was over expressed and the meal size was small, the feeding frequency was indicating direct impact on foraging behavior by increasing it.
8.a) 8.b)
Control dAP2 -
CG10440 - dAP2 +
dAP2 +; CG10440 - 0
50 100 150 200
________________
| ___________|
| _____|____
| | |
***
**
** *
Number of feeding bouts
Control dAP2 -
CG10440 - dAP2 +
dAP2 +; CG10440 - 0.00
0.01 0.02
0.03 __________*
| |
Volume of meal size (µl)
Figure 8: Effect of AP-2 and CG10440 on foraging behavior. The CAFE assay test was conducted on 5 to 7 days old male flies (n=5) and observed data was plotted against experimental and control strains with mean and SEM. dAP2-: AP-2 knockdown in Tdc2 neuron, CG10040-: CG10440 knockdown in Tdc2 neuron, dAP-2+: AP- 2 over-expressed in Tdc2 neuron and dAP-2+;CG10440-: over expressed AP-2 in the absence of CG10440 in Tdc2 neuron. a) Total number of feeding bouts in 24 hours plotted against experimental and control flies, b) volume of meal size per intake of fly against different strains. Significant difference between the groups:
P<0.05(*); P<0.001(**) and P<0.0001(***).
25
3.4. The effect of AP-2 and CG10440 on feeding after starvation:
The previous experimental results suggested that over expression of AP-2 in octopaminergic neurons increases the food intake of the fly. This method was used to observe feeding behavior after acute starvation; flies were subjected to 18 hours of starvation and were then observed for the feeding behavior by feeding them with two sets of food. The starved flies were given the first set of food without dye for fifteen minutes and for the next fifteen minutes, they were provided with the second set of food with a blue dye. Thus the feeding behavior was measured by analyzing the colour of the gut of the flies; thereby the feeding amount could be determined. All the mutated flies had a significant increase in their craving behavior when compared to control (P<0.05). Over-expression of AP-2 increased craving behavior compared to all flies. However, in the absence of CG10440, over-expression of AP-2 reduced the craving behavior similar to the control (Fig.9) (Table.1).
control dA
P2- CG1
0440- dA
P2+
dAP2+;
CG1 0440- 0
20 40 60
________________
|_____ |
| |
__________
| _____|_____
| | | ***
* *
** ***
% Over-fed flies
Figure.9: Effect of AP-2 and CG10440 on craving behavior after starvation. Flies were starved for 18 hours then feed with normal food for 15 min followed by blue colored food for 15 min. The over-fed flies, which had blue crop, were counted and percentage of over fed flies were plotted. dAP2-: AP-2 knockdown in Tdc2 neuron, CG10040-: CG10440 knockdown in Tdc2 neuron, dAP-2+: AP-2 over-expressed in Tdc2 neuron and dAP- 2+;CG10440-: over expressed AP-2 in the absence of CG10440 in Tdc2 neuron. Significant difference between the groups: P<0.05(*); P<0.001(**) and P<0.0001(***).
Table.1: The effect of genes AP-2 and CG10440 on foraging and feeding behaviour
AP-2+;
Control AP-2- CG10440- AP-2+ CG10440-
Feeding frequency 43.3±6.8 59.7±5.5 62.50±6.8 140.1±23.49 72.7±15.86 Meal size/intake (μl) 0.0144±0.001 0.021±0.003 0.018±0.003 0.0092±0.0008 0.013±0.002 Craving after starvation (%) 3±4 25±17.32 21±11.94 51±10.84 11±3.17
*Values are means ± SE
