The Drosophila PGC-1 homologue Spargel coordinates mitochondrial biogenesis to insulin-signalling

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1 Research Collection Doctoral Thesis The Drosophila PGC-1 homologue Spargel coordinates mitochondrial biogenesis to insulin-signalling Author(s): Tiefenböck, Stefanie Katharina Publication Date: 2009 Permanent Link: Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library

2 DISS. ETH NO THE DROSOPHILA PGC-1 HOMOLOGUE SPARGEL COORDINATES MITOCHONDRIAL BIOGENESIS TO INSULIN-SIGNALLING A dissertation submitted to ETH ZURICH for the degree of Doctor of Sciences presented by STEFANIE KATHARINA TIEFENBÖCK Mag. rer. nat., University of Vienna 29. Oktober 1980 Austria Accepted on the recommendation of Prof. Dr. Christian Frei Prof. Dr. Wilhelm Krek Prof. Dr. Pierre Léopold Prof. Dr. Walter Wahli 2009

3 INDEX ABSTRACT 3 ZUSAMMENFASSUNG 4 1. INTRODUCTION Mitochondria and the regulation of mitochondrial biogenesis The PGC-1 family of transcriptional coactivators Mitochondrial biogenesis in the fly Aim of the study Significance RESULTS 2.1. Drosophila Spargel is the only fly homologue of the mammalian PGC-1 family of transcriptional coactivators Phenotypic characterization of the spargel mutant and rescue of these phenotypes Spargel is required for normal larval development and growth Rescue of the spargel mutant phenotypes by a genomic rescue construct and a UAS-Srl transgene Spargel mrna is expressed in may larval tissues Cellular phenotypes of the spargel mutant Cell-autonomous versus non-autonomous effects in the spargel mutant Spargel is required for cell-autonomous growth and survival Spargel overexpression leads to reduced cellular size Microarray analysis of the spargel mutant fat body The effect of Spargel on mitochondrial biogenesis and its in vivo interaction with Delg, the homologue of mammalian NRF-2α Spargel and Delg share many target genes Spargel and Delg function in parallel pathways in respect to mitochondrial mass 37 Stefanie Katharina Tiefenböck 1

4 Oxidative phosphorylation defects in the spargel delg double mutant Discussion Spargel is required for insulin-signalling mediated growth and controls part of the transcriptional changes induced by insulin-signalling Background Spargel is required for insulin-signalling mediated cellular growth Spargel mediates transcription in response to insulin-signalling (microarray analysis) The activation of insulin receptor leads to increased mitochondrial biogenesis Insulin signaling leads to increased Spargel protein levels Spargel mediates a negative-feedback on insulin-signalling DISCUSSION AND OUTLOOK APPENDIX Summary of microarray data on all nuclear encoded mitochondrial proteins GO Term enrichment analysis of INR-overexpression data and the dependence on Spargel MATERIALS AND METHODS ACKNOWLEDGEMENTS CURRICULUM VITAE REFERENCES 85 Stefanie Katharina Tiefenböck 2

5 ABSTRACT Mitochondrial biogenesis must be adapted to tissue function, cell proliferation and growth, and nutrient availability. In mammals, the related transcriptional coactivators PGC-1α, PGC- 1β and PRC regulate multiple metabolic functions, including mitochondrial biogenesis. However, we know relatively little about their respective roles in vivo. Here we show that the Drosophila PGC-1 family homologue Spargel promotes the expression of multiple genes encoding mitochondrial proteins. Spargel was not limiting for mitochondrial mass and OXPHOS activity and functions in parallel to Delg, the fly NRF-2α/GABPα homologue. Accordingly, the spargel delg double mutant showed strongly exacerbated mitochondrial defects compared to single mutants. More importantly, in the larval fat body, Spargel mediated mitochondrial biogenesis, cell growth and the transcriptional control of target genes in response to insulin-signalling. In this process, Spargel functioned in parallel to the insulinresponsive transcription factor dfoxo and provided a negative feedback loop to fine-tune insulin-signalling. Together, our data place Spargel at a nodal point for the integration of mitochondrial biogenesis to tissue and organismal metabolism and growth. Stefanie Katharina Tiefenböck 3

6 ZUSAMMENFASSUNG Mitochondrielle Biogenese ist essentiell für die Anpassung des Energiehaushalts einer Zelle an entwicklungs- und gewebsspezifische Veränderungen, sowie an vorherrschende Umweltbedingungen (zum Beispiel Sauerstoff- und Nährstoffverfügbarkeit oder Temperaturschwankungen). In Wirbeltieren wird dieser kritische Prozess durch eine Familie von transkriptionellen Kofaktoren, PGC-1α, PGC-1β und PRC reguliert, die neben ihrem Effekt auf mitochondrielle Biogenese eine Vielzahl anderer Stoffwechselprozesse (unter anderem Glucose- und Fettmetabolismus) kontrollieren. Trotz dieser wichtigen Funktionen weiss man zum jetzigen Zeitpunkt relativ wenig über die in vivo Funktion dieser Proteine. Diese Studie beschreibt das funktionelle Homolog von PGC-1 Proteinen in der Fruchtfliege Drosophila melanogaster, genannt Spargel, und dokumentiert den Effekt dieses Proteins auf mitochondrielle Biogenese in der Fliege. Wir zeigen, dass Spargel die Expression einer Vielzahl nukleär kodierter mitochondrieller Proteine reguliert und zwar in einem parallelen Signalweg zu Drosophila Delg, ein Homolog des menschlichen NRF-2α. Ausserdem zeigen wir, dass Spargel den molekularen Zusammenhang zwischen mitochondrieller Biogenese und dem Insulin-Signalweg herstellt und somit mitochondrielle Funktionen mit Zellwachstum koordiniert. Stefanie Katharina Tiefenböck 4

7 1. INTRODUCTION During development of an organism, growth (accumulation of mass) and proliferation (cell cycle progression) are highly regulated. These two processes are often linked but they are separate and controlled by different mechanisms. These mechanisms ensure that tissues or organisms grow to a certain size, both in response to environmental stimuli and genetic factors. Whereas cell cycle progression is clearly defined, accumulation of mass can be achieved by several mechanisms: synthesis of proteins, lipids or carbohydrates or changes in the assimilation of nutrients. As growth and the adaptation to environmental changes are highly dependent on an accurately regulated energy household, the regulation of metabolism plays a major role. Mitochondria are cellular organelles that serve critical functions in the regulation of energy homeostasis, metabolic pathways, cell signalling and survival. An essential function of mitochondria is the generation of energy out of nutrients such as carbohydrates, lipids and amino acids. Thus one would expect the coordination of mitochondrial mass and activity with cellular growth rates. Since there is only little known about the regulation of mitochondria in response to growthpromoting pathways and nutrient availability, the main interest of my PhD thesis was to elucidate the in vivo regulation of this process. Many growth-signalling pathways have been studied in detail in the fruit fly Drosophila melanogaster making this organism ideal to study the in vivo connection between growth control and mitochondrial biogenesis. Stefanie Katharina Tiefenböck 5

8 1.1. Mitochondria and the regulation of mitochondrial biogenesis Mitochondria are cytoplasmic organelles that constitute a hall-mark of eukaryotic cells. They are comprised of a soluble matrix surrounded by a double-membrane, an ion-impermeable inner membrane and a permeable outer membrane. This specific architecture of the mitochondrion allows a number of vital cellular processes to take place, the main function being the generation of energy from nutrients through the production of ATP by oxidative phosphorylation. In addition, mitochondria generate metabolites used for anabolic processes (lipid- and protein metabolism) and serve other critical functions in the adaptation to physiological changes and cell survival. Thus, it is not surprising that defects in mitochondrial number and function are associated with a broad spectrum of pathologies, such as neurodegenerative diseases, diabetes, aging and cancer (reviewed in Wallace, 2005). A key in the normal control of mitochondrial function is the regulation of mitochondrial biogenesis. Mitochondria can not be formed de novo, but emerge from preexisting mitochondria. Mitochondria have their own DNA (mtdna) encoding some proteins of the oxidative phosphorylation (OXPHOS), as well as trnas and rrnas. The maintenance, replication and transcription of the mitochondrial genome is controlled by a wellcharacterized mitochondrial-specific transcription complex: consisting of the mitochondrial RNA polymerase POLRMT, the transcription factor Tfam and two mitochondrial transcription specificity factors, TFB1M and TFB2M. All these factors are encoded in the nucleus like the majority of mitochondrial proteins. They are imported into the mitochondrion post-translationally, thereby increasing mitochondrial mass. Thus the tight coordination of the expression programs between these two compartments is essential for mitochondrial biogenesis (Kelly and Scarpulla, 2004; Ryan and Hoogenraad, 2007; Scarpulla, 2008). In Stefanie Katharina Tiefenböck 6

9 addition, the plasticity of mitochondria must be adapted to environmental conditions, such as tissue function, cell proliferation and growth, and nutrient availability The PGC-1 family of transcriptional coactivators A major regulator in the nuclear control of mitochondrial biogenesis in response to environmental stimuli (like low ATP, growth hormones, NO, high Ca 2+ (reviewed by Clementi and Nisoli, 2005; Kelly and Scarpulla, 2004) is the family of PGC-1 proteins. It consists of three family members: PGC-1a (PPARγ coactivator 1), the founding member of this family (Puigserver et al., 1998), PGC-1β (Kressler et al., 2002; Lin et al., 2002a) and PRC (PGC-1 related coactivator) (Andersson and Scarpulla, 2001). The PGC-1 proteins are highly versatile transcriptional coactivators that regulate a number of different metabolic processes by binding and coactivating different nuclear receptors and transcription factors in a tissue- and stimulus-specific way: PGC1α, for example, was first identified as a potent inducer of adaptive thermogenesis upon cold-stimulation in brown adipose tissue by coactivation of PPARγ (Puigserver et al., 1998). In the liver, it interacts with FOXO1 (Puigserver et al., 2003) or HNF4α (Yoon et al., 2001) to induce gluconeogenesis upon starvation and it can control differentiation programs such as the fiber-type switching in muscle cells upon exercise by the activation of Mef2 (Lin et al., 2002b). This is by far not a complete list of all described functions and binding partners of the PGC-1 family, a more detailed description of all metabolic functions can be found in recent reviews, such as Finck and Kelly, 2006 and Lin et al., Important for our study, all three members were shown to be potent inducers of mitochondrial mass and function: PGC-1α drives mitochondrial biogenesis by the coactivation of nuclear transcription factors, including NRF-1, NRF-2 (Evans and Scarpulla, 1989; Virbasius and Stefanie Katharina Tiefenböck 7

10 Scarpulla, 1991) and estrogen-related receptor α (ERRα) (Mootha et al., 2004; Schreiber et al., 2004), to enhance the expression of genes encoding mitochondrial proteins (Scarpulla, 2008). These include proteins that are involved in mitochondrial function (respiratory chain complexes, TCA cycle), but also proteins that direct the replication and transcription of the mitochondrial DNA, like TFB1M and TFB2M, as well as the mitochondrial transcription factor Tfam (Gleyzer et al., 2005; Wu et al., 1999). Accordingly, NRF-1 and ERRα are known to be functionally important for PGC-1s to stimulate mitochondria (Mootha et al., 2004; Schreiber et al., 2004; Wu et al., 1999). In addition to enhancing the activity of these transcription factors, PGC-1α overexpression was shown to increase expression levels of NRF-1 and NRF-2α mrna (Wu et al., 1999). However, this tremendous upregulation of NRF-1 and NRF-2α mrna upon PGC-1α induction could not be confirmed by another group (Gleyzer et al., 2005). The reason for this discrepancy is unknown. PGC-1β, a close relative of PGC-1α, also functions as a NRF-1 coactivator (Lin et al., 2002a). The third family member, PGC-1 related coactivator (PRC), exhibits the properties of a cell growth regulator. Like PGC-1α, PRC binds NRF-1 and directs the expression of NRF-1 target genes related to the respiratory chain expression (Andersson and Scarpulla, 2001; Gleyzer et al., 2005). Similarly, NRF-2 promoter binding sites are required for coactivation by PGC-1α and PRC on certain genes (Gleyzer et al., 2005), and PRC can coactivate NRF-2β (Vercauteren et al., 2008). However, a direct interaction of these two cofactors with NRF-2 has never been shown, suggesting that a third factor is required for the coactivation of NRF-2 through PGC- 1α or PRC. Recent studies showed that PRC complexes with the host cell factor-1 (HCF-1) and NRF-2β, building a functional transcriptional activation complex (Vercauteren et al., 2008). Taken together, these data indicate that PGC-1s and NRF-2 function together on transcription of nuclear encoded mitochondrial proteins. However, it is not known whether NRF-2 is functionally required for PGC-1 s effect on mitochondria, or whether NRF-2 is controlled via other factors. As there is no in vivo data on the interaction of NRF-1/2 and Stefanie Katharina Tiefenböck 8

11 PGC-1 proteins, genetic studies in invertebrates could help elucidate their function in mitochondrial biogenesis. In addition, the redundant functions of PGC-1 proteins on mitochondrial biogenesis have complicated the analysis of their role in basal mitochondrial biogenesis: recently, PGC1α (Leone et al., 2005; Lin et al., 2004) and PGC1β (Lelliott et al., 2006; Sonoda et al., 2007) knock-out mice have been described. Importantly, although these knock-out animals showed reduced expression of multiple genes encoding mitochondrial proteins, mitochondrial mass was either not or only modestly reduced, depending on the tissue (Lelliott et al., 2006; Leone et al., 2005; Lin et al., 2004; Sonoda et al., 2007). In contrast, RNAi-mediated downregulation of PGC-1β in a PGC-1α -/- background led to strong mitochondrial biogenesis defects during the differentiation of brown adipose tissue (Uldry et al., 2006). Furthermore, PGC-1αβ -/- double knockout mice die shortly after birth having strong defects in heart maturation and severe abnormalities in brown adipose tissue function and mitochondrial density (Lai et al., 2008). Therefore, the mild phenotypes observed in single knockouts could be due to redundancy. Alternatively, PGC-1 proteins could be required in tissues that have a large stimulus-induced increase in mitochondrial mass, e.g. during brown adipose tissue differentiation (Uldry et al., 2006) or muscle-type switching upon exercise (Lin et al., 2002b), but otherwise be dispensable for basal mitochondrial mass in most tissues. As there is less or even no redundancy in Drosophila, the fly is an ideal model system to study the function of PGC-1 proteins in the control of basal mitochondrial levels. As mentioned above, mitochondrial biogenesis must be adapted to tissue function, cell proliferation and growth, and nutrient availability. However, relatively little is known about the coordination of mitochondrial biogenesis in the context of growth. Although there is a number of well-described growth driving pathways, including the insulin/tor pathway, the Stefanie Katharina Tiefenböck 9

12 Hippo tumour-suppressor pathway or Myc, our understanding is very limited if and how these growth pathways functionally interact with PGC-1 proteins, and if this in turn controls mitochondrial functions. As most of these growth pathways are well-conserved in the fly, this could be easily addressed in Drosophila. In addition, in the fly a clear requirement of mitochondrial function for CyclinD/Cdk4-driven growth has been described (Frei et al., 2005) Mitochondrial biogenesis in the fly The structure and gene content of the Drosophila melanogaster mitochondrial genome is similar to that of mammals (Garesse and Kaguni, 2005). Analogously, most mitochondrial proteins are encoded in the nucleus and functional homologs of the major factors that control mitochondrial function are described: the mitochondrial DNA Polymerase Polγ (Wernette et al., 1988), the mtssb (Stroumbakis et al., 1994), Tfam (Goto et al., 2001) as well as mttfb1 (Matsushima et al., 2005) and mttfb2 (Matsushima et al., 2004). However, there are no clear Drosophila homologues of PPAR nuclear receptors (King-Jones and Thummel, 2005) and a role for the fly homolog of ERRα in mitochondrial biogenesis not been described. The fly homolog of mammalian NRF-1, Erect wing (ewg), has been studied in the context of early muscle cell and neuronal development but with no described effect on mitochondrial gene expression (DeSimone et al., 1996; DeSimone and White, 1993). The same is true for a described homolog of mammalian NRF-2α, Delg, which was first described for its role in oogenesis (Schulz et al., 1993a; Schulz et al., 1993b). Although based on sequence homology there is a putative homologue of the PGC-1 proteins, CG9809, it has not been functionally characterized. Thus it is not known how the transcriptional control of mitochondrial biogenesis is regulated in the fly. Stefanie Katharina Tiefenböck 10

13 1.4. Aim of the study As the aim of this study was to elucidate how mitochondrial function is controlled in response to nutrients, so we first asked whether a similar transcriptional network depending on PGC1 does exist in invertebrates. While my study concentrates on the characterization of the Drosophila homolog of mammalian PGC-1, a second project in the lab has characterized the role of Delg, the fly homolog of mammalian NRF-2α, in mitochondrial biogenesis (Baltzer et al., submitted). Thus, we were able to also analyze the in vivo interaction of these two proteins in the fly. Second, since many of the growth driving pathways mentioned above are well described in the fly (Eilers and Eisenman, 2008; Grewal, 2008; Saucedo and Edgar, 2007), we investigated whether this putative PGC-1 homolog could mediate the molecular link between growth signals and mitochondrial biogenesis and furthermore if this link is critical for cellular and organismal growth Significance We showed that the Drosophila melanogaster genome encodes a single PGC-1 homologue, Spargel/CG9809, thus providing a system where PGC-1 function can be analyzed without interfering redundancy. Although this homology has been published in the meantime by another group (Gershman et al., 2007), this is the first functional analysis of the Spargel/PGC- 1 mutant phenotype in the fly. In addition, we investigated the role of Spargel in the control of mitochondrial biogenesis in Drosophila and how it is functionally linked with the fly NRF-2α homologue Delg (Baltzer et al., submitted). Moreover, we addressed the question how insulinsignalling affects mitochondria, and found that Spargel was required to a large extent for the transcriptional control in response to insulin-signalling, including for genes encoding mitochondrial proteins. Thus, our data demonstrate a critical role for Spargel/dPGC in the coordination of mitochondria with nutrients and growth. Stefanie Katharina Tiefenböck 11

14 2. RESULTS 2.1. Drosophila Spargel (CG9809) is the only fly homologue of the mammalian PGC-1 family of transcriptional coactivators The fly genome encodes only one putative homolog of mammalian PGC-1 proteins Using blast search, we and others (Gershman et al., 2007) have identified only one PGC-1 homologue in the Drosophila melanogaster genome. This gene, CG9809, is encoded on the right arm of the third chromosome (3R; 82B1) and has two predicted isoforms: CG9809-B and CG9809-D (Fig. 1). These two isoforms are splice-variants of the same transcript and encode a protein of 1067aa (CG9809-PB) or 1058aa (CG9809-PD), respectively. The shorter protein, CG9809-PD, misses 27 nucleotides at the start of exon 3 and has a shorter predicted 3` untranslated region (UTR). Figure 1. Representation of the Spargel (CG9809) locus. Shown is the genomic map of CG9809 (modified from: Flybase, Cytolocation: 3R:248, ,051 [-]. Orange boxes represent coding regions of the respective genes (CG9809, CG31525 or eif3-s10) including 5` untranslated regions (UTR). Grey boxes indicate 3` UTR. The P-element insertion sites of srl 1 and srl 2 loss-of-function alleles are indicated as blue triangles. The genomic rescue construct (Srl GR ; 8700bp) is represented as hatched bar. Both isoforms exhibit 68% and 52% homology with the mammalian PGC-1α and PGC-1β, respectively, in the C-terminus. In addition, many of the functionally important domains are Stefanie Katharina Tiefenböck 12

15 conserved: the N-terminal acidic domain that serves as an interaction platform for the binding of transcription factors (Puigserver et al., 1999), as well as the C-terminal arginine/serine rich domain and the RNA-recognition motif (Fig. 2). The two latter are characteristic for RNA splicing factors and were shown to directly couple the transcriptional activation function of PGC-1 proteins to mrna processing (Monsalve et al., 2000). Interestingly, CG9809 lacks the canonical LXXLL motifs, which for mammalian PGC-1s is required for binding to multiple nuclear hormone receptors and transcription factors. CG9809, however, contains a conserved C-terminal FXXLL motif (Gershman et al., 2007), which could mediate transcription factor binding (Huang et al., 1998). Figure 2. Domains of Drosophila Spargel (CG9809) that are in common with human PGC-1α and PGC-1β. Numbers indicate the respective percentage of homology between the proteins. Acidic: N-terminal acidic domain, RS: arginine/serine-rich domain, RRM: RNA recognition motif. Taken from Gershman et al., Given the high similarity of the protein structure between mammals and the fly, we further investigated whether CG9809 is also a functional homolog of the mammalian PGC-1 proteins. Stefanie Katharina Tiefenböck 13

16 2.2. Phenotypic characterization of the spargel mutant and the rescue of these phenotypes The Drosophila PGC-1 homologue Spargel (CG9809) is required for normal larval development and growth To test whether CG9809 is a functional PGC-1 homologue in flies, we analyzed mutants that have a P-element insertion (KG08646; 11.4kb) into the 5 UTR (Fig. 1). Due to the size of the P-element, such insertions are known to interfere with the normal transcriptional regulation and thus lead to a downregulation of the affected gene. Compared to genetically matched controls (precise excision of the P-element) homozygous mutant larvae have a strong reduction in CG9809 mrna levels (Fig. 3A). In addition, homozygous mutant animals have a defect in larval development: when we followed the increase in larval volume over time (Fig. 3C), we observed that homozygous mutant larvae are delayed in growth starting from the 3 rd instar and reach pupation with a delay by one day. This resulted in significantly smaller pupa (~25% smaller than controls; Fig. 3D). Adults are viable, yet eclose at a lower Mendelian ratio as expected (only 1/6 th is homozygous mutant, instead of 1/3 rd ) and females are sterile. Importantly, the adult flies show a lean phenotype: while the body weight is reduced by ~25% (Fig. 3B), adult body structures, like wings and legs, are almost normal sized (Fig. 3E-G). In addition, the determination of trichome number per wing area did not show any difference between the mutants and the wildtype, demonstrating no change in cell number and cell size (Fig. 3H). To stress the lean phenotype, we termed CG9809 Spargel, German for asparagus, and the KG08646 allele as srl 1. In addition to this allele, we tested another P-element insertion, d04518 (7.3kb), termed srl 2 (Fig. 1). This P-element is inserted at the same position as KG08646 and shows a comparable Stefanie Katharina Tiefenböck 14

17 downregulation in the Spargel mrna (Fig. 3A). The larval and adult phenotypes of the srl 2 mutants are the same as for the srl 1 mutants (Fig. 3B) and data not shown). As ~25% of srl transcript was still detectable in both, srl 1 and srl 2 (Fig. 3A), we tested a transheterozygous combination of srl 1 with Df(3R)ED5046, a deficiency that deletes the Spargel locus as well as neighbouring genes. This led to a further reduction in Spargel transcript levels (Fig. 3A), yet it did not lead to a further decrease in adult weight compared to srl 1 homozygous mutant animals (Fig. 3B). This suggests that srl 1 is at least a strong hypomorphic allele. Figure 3. Spargel is required for larval development and growth. (A) Spargel transcript levels were determined by qrt-pcr. mrna was isolated from whole, mid-third instar larvae: +/+: 4dAED; srl 1/1,srl 2/2 and srl 1 /Df(3R)5046: 5dAED. Expression was normalized to Actin5C (CG4027) and +/+ was set to 1. (B) Wet weight from adult males, genotypes as indicated. Adult males were taken 2 days after eclosion. Number of flies/genotype 10. (C) Shown are larval volumes of wildtype and srl 1 mutants. Pictures of larvae at the indicated time points were taken and larval volumes were determined by measuring the larval dimensions in Adobe Photoshop and subsequent calculation in Excel using the following formula: 4/3π(Length/2) 2 (Diameter/2). Spargel mutants pupate (indicated as P ) at a reduced size with a 1-day delay. (D) Shown are pictures of wildtype and spargel mutant pupae. Pupae were imaged with a 1.6x magnification and Stefanie Katharina Tiefenböck 15

18 pupal volume was calculated as described above for the larval volume. (E) Picture of wildtype and spargel mutant adult males. (F) Femur length of the adult leg. (G) Adult wings were imaged and pixel number per wing area was determined. (H) Trichome number was counted in a defined area of the wing (700x700pixel). For (D) to (H) Size measurements were done by using Adobe Photoshop; n >15. *** equals P<0.001; ** equals P<0.01; * equals P<0.05; ns: not significant The spargel mutant phenotypes can be rescued by a genomic rescue construct and a full-length UAS-Srl transgene To test if the observed phenotypes are specific for the mutation in Spargel and do not result from a secondary mutation in the genome, we created transgenic flies carrying a genomic rescue construct (Srl GR ; 8.7kb; Fig. 1). Although the spargel mrna levels are only partially restored, this line rescues all mutant phenotypes including the defect in larval development and the reduced adult body weight (Fig. 4A-C). It also rescues the phenotypes of the transheterozygous combination of srl 1 with Df(3R)ED5046 (data not shown). These results show that the observed phenotypes are specific for a mutation in the Spargel locus. Figure 4. Rescue of the spargel mutant phenotypes. (A) Spargel transcript levels were determined by qrt- PCR. mrna was isolated from whole, mid-third instar larvae (srl 1/1 : 5d AED; all others: 4d AED). Expression was normalized to Actin5C (CG4027) and +/+ was set to 1. (B) Complete rescue of the reduced weight phenotype of adult srl 1/1 mutants by the genomic rescue construct, Srl GR. (C) Srl GR rescues the larval size defect Stefanie Katharina Tiefenböck 16

19 and the delay in larval development. Shown are larval volumes of the indicated phenotypes. P indicates the start of pupation. (D) Rescue of the reduced adult body weight by hs-gal4 driven UAS-Spargel (UAS-Srl) with one 1.5h heat shock/day (done by Ch. Frei). (E) CG31525 mrna was isolated from mid-3 rd instar fat bodies, quantified by qrt-pcr and normalized to gammatub23c (CG3157). For (B) and (D): For all weight measurements, adult males were taken 2 days after eclosion. Number of flies/genotype 10. *** equals P<0.001; ns: not significant. As another gene, CG31525, is fully encoded within the Spargel locus (Fig. 1) and since the genomic rescue construct includes this gene, we also created a fly line expressing specifically a full-length Spargel cdna (including 5` and 3`UTRs) under the control of the UAS promoter. When driven using heat-shock Gal4, UAS-Srl also suppressed the mutant phenotypes (Fig. 4D and data not shown), except for the female sterility. This can be explained by the fact that the vector used to create this construct, puast, is not expressed in the germline. In addition, the CG31525 mrna levels are not affected in the srl 1 mutant (Fig. 4E). This demonstrates that Spargel, and not CG31525, is responsible for the observed phenotypes Spargel mrna is expressed in many larval tissues To continue with the characterization of cellular phenotypes caused by the spargel mutant, we first analyzed the temporal and spatial expression pattern of Spargel in wildtype animals using qrt-pcr. Searching FlyExpress, an available internet database for gene expression data, we found that Spargel mrna is expressed at different embryonic stages as shown by in situ hybridization studies (Reference: Van Emden B, Ramos H, Panchanathan S, Newfeld S, and Kumar S (2006). FlyExpress: An image-matching web-tool for finding genes with overlapping patterns Stefanie Katharina Tiefenböck 17

20 of expression in Drosophila embryos. ( Arizona State University, Tempe, Arizona , USA.). To get more detailed information about the expression pattern of Spargel, we performed qrt-pcr on whole animals at different developmental stages. Our data showed that Spargel in general is expressed at relatively low levels (Fig. 5A, normalized to the Act5C mrna). Starting with a modest expression at early 3 rd instar, Spargel mrna further decreases during larval development. While its expression is still relatively low in the pupa, Spargel reaches an expression maximum in the adult female. Data retrieved from FlyAtlas (Chintapalli et al., 2007) ( confirm this expression pattern and show that Spargel mrna is significantly enriched in the ovaries. As srl 1 mutant female flies are sterile, this could indicate a role of Spargel in oogenesis, yet we have not further tested this. As we are especially interested in the growth period of the feeding animal and as srl 1 mutant animals show a strong phenotype already at larval stages (retarded growth and delayed development), we looked at the tissue-specific expression during the mid-3 rd instar. For this, we dissected out different tissues from wildtype larvae (4d AED), including the fat body, gut, salivary glands and brain. In this experiment we used Rp49 as normalization control because its expression levels were comparable between the different larval tissues (in contrast to Act5C). Spargel mrna is expressed in all these tissues: the fat body and the salivary glands had comparable expression levels of Spargel, gut and muscle showed a reduced expression (Fig. 5B). Importantly, the Spargel mrna is highly expressed in the larval brain. Although this result might reflect an important function for Spargel within the central nervous system, similar as its mammalian homologs (Lin et al., 2004), the functional significance of this finding remains to be determined. Stefanie Katharina Tiefenböck 18

21 Figure 5. Spargel expression pattern during larval development and in different larval tissues. For (A) and (B): Spargel levels were detected by qrt-pcr and normalized to Act5C or Rp49. (A) Animals were taken at the indicated developmental stage. 3 rd instars: early: 3d AED; mid: 4d AED; late: 5d AED; Pupa: 3d after puparium formation; Females: 1d after eclosion (note that this is the result of a single experiment). (B) Tissues were dissected from mid-3 rd instar wildtype controls. Shown are the results of two biological replicates. Number of tissue/experiment >25. Stefanie Katharina Tiefenböck 19

22 Spargel mutant fat body cells have changed morphology due to big lipid droplets, but only minor defects in mitochondrial mass Having observed the defects in larval development and on organismal size (Chapter ), we next asked if we can observe cellular phenotypes that are caused by the srl 1 allele and whether these would account for the defects in animal growth. For the following analyses, we decided to focus on the larval fat body for several reasons: - First, we found that among the larval tissues, except for the brain, Spargel expression levels are the highest in the fat body. This is interesting because also the mammalian PGC-1 proteins were shown to play a crucial role in adipose tissue development and function (Puigserver et al., 1998; Uldry et al., 2006). - Second, the fat body plays an important role in the regulation of larval growth: as an adipose tissue that also exerts liver-like functions, it regulates nutrient storage and release and thus controls the organismal energy supply during the larval growth period (reviewed in (Leopold and Perrimon, 2007). In addition, the fat body was shown to act as an endocrine tissue that controls the growth of imaginal discs by releasing growth hormones (Kawamura et al., 1999). - Third, the larval fat body is an endoreplicative tissue that consists of a monolayer of large cells. This makes it relatively easy to dissect, perform immunofluorescence and obtain enough material for a variety of experiments from even a low number of animals. As mentioned in the Introduction (Chapter 1.2.), mammalian PGC-1 proteins were shown to be critical in the regulation of mitochondrial biogenesis (Scarpulla, 2008). To test whether mitochondria are affected in the srl 1 mutant, we performed a first experiment to monitor mitochondrial number in the larval fat body using MitoTracker, a mitochondrial-specific dye. In control animals, mitochondrial staining is abundant throughout the cytoplasm (Fig. 6). In Stefanie Katharina Tiefenböck 20

23 spargel mutants, we detected only a minimal reduction in staining. However, this could be due to a change in the morphology of the fat body cells in the mutant (discussed below). Thus, using MitoTracker we can not provide strong evidence for an effect on mitochondrial mass and additional experiments are required to analyze this more in detail. Moreover, the MitoTracker staining cannot monitor defects in mitochondrial functions like enzymatic activities or the respiratory capacity. Therefore we used other read-outs to perform a more detailed analysis of the effect of Spargel on mitochondrial biogenesis. The results of these experiments are presented and discussed in the Chapters 2.4. and 2.5. Figure 6. Mitochondria-specific MitoTracker stainings of larval fat bodies. Age: mid-3 rd instar; +/+: 4d AED, srl 1/1 : 5d AED. DAPI stains the nuclei. Bar equals 20µm. As mentioned above, during the MitoTracker experiments we observed a striking change in the morphology of the spargel mutant fat body cells: the cytoplasm is filled with droplet-like structures giving them an empty appearance compared to the wildtype control (Fig. 6). A main function of the fat body is the storage of excess dietary fat in the form of lipid droplets. To determine if the observed structures are lipid droplets we performed a Nile red staining Stefanie Katharina Tiefenböck 21

24 which specifically stains intracellular lipid stores. As shown in Figure 7A. the lipid droplets in wildtype cells are small and numerous. In the spargel mutant fat body, however, lipid droplet size is strongly increased. In addition, these big lipid droplets can be found throughout the cytoplasm of the mutant fat body cells which is shown in the pictures of different cell layers taken by differential interference contrast (DIC) microscopy (Fig. 7B). B Section 1 Section 2 Section 3 +/+, section 1 +/+, section 2 +/+, section 3 srl 1/1, 6d AED +/+, 5d AED srl 1 /srl 1, section 1 srl 1 /srl 1, section 2 srl 1 /srl 1, section 3 Figure 7. Spargel mutants have big lipid droplets. (A) Nile red (lipids) and DAPI (nuclei) staining of mid-3 rd instar fat body. Genotypes as indicated. (B) DIC images of different sections on late 3 rd instar fat body. Arrows Stefanie Katharina Tiefenböck 22

25 indicate the position of the nuclei. Arrowheads mark lipid droplets. For (A) and (B): Scale bars correspond to 20µm. Furthermore, the observed lipid droplet phenotype can be reversed by the genomic rescue construct, Srl GR (Fig. 7A). These findings suggest a role for Spargel in the control of lipid metabolism and we looked into this more in detail. Big lipid droplet phenotypes were previously described in the fat body cells of larvae kept under low nutrient conditions (Zhang et al., 2000; Colombani et al., 2003). It is believed that the change in lipid droplet morphology helps to mobilize the fat stores by making them accessible to the TAG lipase Brummer (Gronke et al., 2005), therefore providing other tissues with energy. Thus, the spargel mutant phenotype could result from a systemic, starvation-like effect due to the lack of Spargel. To investigate if lipid stores are used up to a higher extent in the mutants, we analyzed the total organismal fat contents. For this, we took feeding whole mid-3 rd instar larvae and determined the amount of total triacylglyceride (TAG), the storage form of fat. When normalized to total body protein or total body weight, we did not observe any change in the total triacylglyceride (TAG) levels in the spargel mutants relative to the wildtype (Fig. 8). Thus the lipid droplet phenotype in the fat body does not reflect a depletion of organismal lipid contents, however, it is still possible that the phenotype results from a direct effect of Spargel on lipid homeostasis, such as lipid remobilization and distribution. Stefanie Katharina Tiefenböck 23

26 Figure 8. Spargel mutants have normal amounts of total body fat. TAG levels of feeding mid-3 rd instar larvae, normalized to total body weight or total protein. Shown are the results of three biological replicates. Kindly provided by Nicole Egli. Recently, Gutierrez and colleagues have described the oenocytes as major regulators of those processes (Gutierrez et al., 2007). Oenocytes are clusters of specialized cells with hepatocytelike function that are found in the body wall of larvae and adults. Under fed conditions these cells do not contain any lipids. Upon starvation, however, lipids are mobilized from the fat body and accumulate in the oenocytes where they are processed for subsequent redistribution to other tissues. To see if the spargel mutant oenocytes ectopically accumulate lipids even under fed conditions, we stained the body wall of feeding mid-3 rd instar larvae with Nile red. Unfortunately, in our hands, we detected lipid accumulations already in the feeding wildtype controls (data not shown). This is probably due to a different fly food composition used in our lab. As our assays did not allow us to detect any changes in total lipid content or distribution, we decided to investigate the expression response of genes involved in lipid metabolism more in detail by microarrays (see Chapter 2.4.). As we so far only investigated the phenotypes of whole spargel mutant animals, we can not distinguish if the observed cellular defects in the fat body are of cell-autonomous or non- Stefanie Katharina Tiefenböck 24

27 autonomous origin. This question is addressed by the experiments described in the next chapter Cell-autonomous versus non-autonomous effects in the spargel mutant As described before, we detected high levels of Spargel mrna in the brain, therefore the observed mutant phenotypes in the fat body could be due to a systemic effect (e.g. through the release of hormones). Alternatively, Spargel could be required in a cell-autonomous manner in the fat body. To look into this more in detail, we induced spargel loss-of-function clones in the fat body, and tested for mutant phenotypes Spargel is required for the cell-autonomous control of growth and survival To analyze the cell-autonomous effect of the srl 1 mutation, we used the Flp/FRT system (Golic and Lindquist, 1989). Due to the location of Spargel on the chromosome (82B1), it was not feasible to induce Spargel loss-of-function clones by the recombination of srl 1 onto the FRT82-chromosome. Thus, we took a reverse approach and recombined an insertion of Srl GR (genomic rescue) on the 2 nd chromosome (chromosome arm: 2R) onto the FRT42- chromosome, and analyzed flies that are mutant for Spargel (which is located on the third chromosome). The resulting clones are either homozygous mutant for Spargel (GFP positive) or wildtype (GFP negative) due to the presence of two copies of Srl GR (Fig. 9D). Stefanie Katharina Tiefenböck 25

28 Figure 9. Spargel is required for cell-autonomous growth and survival. (A) Phalloidin and DAPI staining of mid-3 rd instar fat body. Clones were induced using the Flp/FRT system. Genotype: hs-flp 122 ; FRT42, Ub- GFP/FRT42, srl GR ; srl 1 /srl 1. GFP-/- cells are wildtype (marked by arrows with open ending), GFP+/- cells are heterozygous for srl 1 and GFP+/+ cells are homozygous srl 1 mutant (marked by arrows with filled arrowhead). (B) Most fat bodies lack srl homozygous mutant twinspot cells. (C) Shown are spargel mutant clones in the wing disc of a mid-l3 larva. Clones were induced 48h after egg deposition. Wildtype and spargel mutant clones are indicated as in (A). (D) Schematic representation of the clone formation in the larval fat body. Clones are induced by a heat shock during mid-embryogenesis (6-8h after egg deposition). After twinspot formation, these cells typically divide once or twice, leading to a clone of 2 or 4 cells, respectively. After this, these cells stop mitotic division, and endoreplicate (S-G cycles without interfering mitosis), leading to a large increase in cell size. For (A) and (B): 20x magnification, scale bar corresponds to 50µm. For (C): 40x magnification, scale bar corresponds to 20µm. As shown in Figure 9A, cellular and nuclear size is decreased in the srl 1 mutant clones, suggesting a cell-autonomous requirement for Spargel during growth. Importantly, in the majority of the analyzed fat body tissues we could not detect the spargel mutant sister cells (Fig. 9B). This could point out a vital function for Spargel in cell survival. Stefanie Katharina Tiefenböck 26

29 As the number of cells per clone in the fat body is limited we aimed to look at the survival of spargel homozygous mutant clones in a mitotic tissue. We chose the wing disc because the number of cells per clone can easily be varied by inducing clone formation at different time points during disc development. Furthermore, it was shown, that cells carrying a mutation that leads to reduced fitness are removed from the disc epithelium by its healthy neighbour cells, a process called cell competition. To test, if spargel mutant cells have a survival defect, we induced Spargel loss-of-function clones in the wing disc (Fig. 9C). Interestingly, we find that similar to the fat body, spargel mutant clones are reduced in size, and this is due to a reduced cell number. Although the process of cell competition has not been described for the fat body, it is most likely that the spargel mutant twin spot cells in the fat body also die and this probably happens very early in fat body development. Considering Spargel`s role in cell survival, the few homozygous mutant clones we obtained in the fat body could result from additional compensatory mutations that help the cells survive the lack of Spargel. This could partially also explain that we could not observe any cellautonomous changes in mitochondrial mass or lipid droplet morphology (data not shown), therefore we cannot fully exclude a direct effect of Spargel on these processes Clonal overexpression of Spargel leads to reduced cell size in the fat body We showed that Spargel is involved in the regulation of cell-autonomous growth and survival by analyzing loss-of-function clones (described above). To determine the effect of increased Spargel levels, we overexpressed UAS-Spargel (UAS-Srl) in random clones in the fat body of wildtype larvae using the hs-flp; Tub>CD2>Gal4, UAS-GFP system (Flip-out/Gal4-system, (Scott et al., 2004). Stefanie Katharina Tiefenböck 27

30 Figure 10. The overexpression of Spargel leads to cell-autonomous growth defects. UAS-Srl was expressed in random clones using the hs-flp; Tub>CD2>Gal4, UAS-GFP system in wildtype background. Shown are stainings from larval fat bodies, using MitoTracker (red) specific for mitochondria, DAPI (blue) specific for DNA and GFP (green), which marks UAS-Srl expressing cells. Bar equals 20µm. Kindly provided by Christian Frei. Compared to the controls, cells overexpressing UAS-Srl are reduced in cell size (Fig. 10). This is surprising, as we demonstrated before that spargel loss-of-function clones are smaller and moreover, spargel mutant larvae display a general growth defect (see Chapter ). We hypothesize that the small size phenotype of cells that overexpress Spargel could result from a dosage effect and that the exact homeostasis of Spargel levels might be critical for the induction of physiological changes. Interestingly, such an effect has not been reported in the mammalian system upon PGC-1α overexpression. This could be due to a weaker induction of the protein in mammals as compared to our set-up. To check if the observed dominant effect on growth by Spargel overexpression can be rescued by a reduction in Spargel levels we could perform the same experiment in a Spargel RNAi background. For this a recently available Spargel RNAi fly line (Vienna Drosophila RNAi Center, Dietzl et al., 2007) could be used. In addition, the size defect prevents a conclusive analysis about the cell-autonomous effect of Spargel overexpression on mitochondrial abundance as the cytoplasmic area is too strongly affected to allow a reliable visualization of mitochondria by MitoTracker staining (Fig. 10). Stefanie Katharina Tiefenböck 28

31 Taken together, the phenotypic characterization of the spargel mutant shows a role for Spargel in the regulation of larval growth and development including a mild mitochondrial defect and a stronger lipid droplet phenotype that is specific for the larval fat body. In addition, the analysis of loss-of-function and gain-of-function clones shows a cellautonomous requirement of Spargel for growth in the larval fat body, whereby Spargel levels seem to be critical. However, by the experiments conducted so far, we can not exclude an additional non-cell-autonomous effect of Spargel. To sort this out, we could test the effects of tissue-specific rescues or knock-downs on the whole organism. Unfortunately, the clonal analysis experiments could not clarify the role of Spargel on mitochondria and lipid metabolism. In order to check the effects of Spargel on metabolic pathways we performed a global analysis of transcriptional changes in the spargel mutant by microarrays (next chapter). Stefanie Katharina Tiefenböck 29

32 2.4. Microarray analysis of the spargel mutant larval fat body reveals a requirement of Spargel for the proper expression of multiple genes involved in mitochondrial function and energy metabolism As discussed in the Introduction the mammalian PGC-1 proteins are a family of transcriptional coregulators that play a key role in the control of different metabolic processes, including lipid and glucose homeostasis, mitochondrial biogenesis and energy homeostasis (Lin et al., 2005). To address a likely Spargel function as a transcriptional regulator, we performed genome-wide microarray analysis using dissected fat bodies. As described in previous experiments, feeding mid-third instar larvae were taken for all experiments (4 days after egg deposition (AED) for control, and 5d AED for spargel mutants). The full data set can be accessed at the NCBI database (Edgar et al., 2002) using accession number GSE GO Biological Process, >1.5x down in srl 1/1 mutant fat body (P<0.05) GO Biological Process, >1.5x up in srl 1/1 mutant fat body (P<0.05) GO ID P-value Term GO ID P-value Term Mitochondrial Biogenesis and Function Developmental process GO: E-25 Electron (e - ) transport GO: E-03 Puparial adhesion GO: E-24 Oxidative phosphorylation GO: E-02 Molting cycle, chitin-based cuticle GO: E-23 ATP synthesis coupled e - transport GO: E-22 Generation of precursor metabolites and energy Lipid metabolism GO: E-14 Mitochondrial electron transport, NADH to ubiquinone GO: E-05 Lipid catabolic process GO: E-03 Mitochondrial transport GO: E-04 Fatty acid beta-oxidation GO: E-02 Mitochondrial organization GO: E-04 Cellular lipid catabolic process GO: E-02 Mitochondrial e - transport, cytochrome c to oxygen Transcription and Translation Others GO: E-49 Translation GO: E-03 Transport GO: E-33 Gene expression GO: E-03 Localization GO: E-15 Protein metabolic process GO: E-30 Ribosome biogenesis and assembly GO: E-18 rrna metabolic process GO: E-03 Protein folding GO: E-04 Nucleobase, nucleoside and nucleotide metabolic process Cell cycle GO: E-18 Spindle elongation GO: E-12 Mitotic spindle organization and biogenesis GO: E-06 Microtubule cytoskeleton organization/biogenesis GO: E-05 Mitotic cell cycle Cellular metabolic processes GO: E-36 Metabolic process GO: E-18 Cellular biosynthetic process GO: E-17 Macromolecule biosynthetic process GO: E-15 Cellular protein metabolic process GO: E-06 Organelle organization and biogenesis Table 1. GO-Term enrichment for genes that were significantly up- or downregulated >1.5-fold in the srl 1/1 mutant fat body compared to wildtype. Shown are the GO terms that are significantly enriched (P-value <0.05). Stefanie Katharina Tiefenböck 30

33 Using Affimetrix chips with probes per array, we detected 8871 probes with a present signal in the fat body. Out of these, a total number of 2827 genes (31.87%) was more than 1.5x regulated in the srl 1 mutant. Among these, the transcript levels of 1019 genes (11.49%) were increased and 1808 genes (20.38%) showed decreased expression. Spargel transcript itself was reduced to 29.38% (Log 2 Ratio: , P-value: ) in the srl 1 mutant fat bodies. To find out which processes are mostly affected by the transcriptional deregulation of these genes, we performed a Gene Ontology (GO) Term analysis. Interestingly, we found only a few GO processes enriched in the spargel mutant that were significantly upregulated and these could be manually clustered into three main GO categories (Table 1): developmental process, lipid metabolism and general signalling and transport events. The upregulation of genes involved in developmental processes, like puparial adhesion and molting cycle, could be a result from the 1-day developmental delay of spargel mutant larvae. Interestingly, many genes involved in lipid metabolism including fatty acid beta-oxidation are upregulated (Table 2). This reflects a major deregulation of lipid metabolism and agrees with the lipid droplet phenotype observed in the spargel mutant fat body (Chapter ). Although at this timepoint we can not fully exclude a direct regulation of certain genes involved in lipid metabolism through Spargel, a secondary effect on lipid metabolism is more likely. Gene Name CG Number Ratio (srl 1/1 vs. Srl wt/wt ) P-Value ß-oxidation CptI CG MCAD CG Thiolase CG CG4388 CG Lipid homeostasis Lsd-1 CG Lsd-2 CG Lipase Bmm CG Table 2. List of selected genes involved in lipid metabolism. Shown are the expression data from the fat body-specific microarray analysis described. The transcript levels of these genes are significantly upregulated in the spargel mutant fat body. The ratio indicates the relative fold change in gene expression between srl 1/1 to control (precise excision). Stefanie Katharina Tiefenböck 31

34 In contrast, the enrichment for GO processes among the downregulated genes revealed a number of different categories that were affected in the spargel mutant (Table 1). Importantly, genes involved in mitochondrial functions, in particular oxidative phosphorylation (OXPHOS; mostly electron transport complexes I and V), were expressed at significantly reduced levels in the spargel mutant. In addition to the deregulation in mitochondria, only a few non-mitochondrial functions were downregulated in the spargel mutant. These include translation, gene expression and RNA biology, processes that are essential for the accumulation of cellular mass. We showed before that spargel mutant larvae have growth defects (2.2.1.), therefore this phenotype probably results from a combination of Spargel s effect on mitochondrial and non-mitochondrial functions. At this point, we will first continue with the discussion of Spargel s role in mitochondrial biology, the effect on organismal growth will be discussed more in detail in the Chapters 2.6. and 3. As one main function of the mammalian PGC-1 proteins is the transcriptional control of nuclear encoded mitochondrial genes (reviewed in (Scarpulla, 2002; Scarpulla, 2008)), we performed a detailed analysis of all predicted 313 nuclear genes encoding mitochondrial proteins (source: MitoDrome, (Sardiello et al., 2003)). Out of 252 detected genes, only 14.68% were upregulated (Table 3; and Appendix, section 4.1.). These included genes involved in stress response (heat-shock proteins) and fatty acid beta-oxidation which has already been discussed above. In contrast to the relatively low number of mitochondrial genes with increased expression, more than half (55.16%) of the detected genes involved with mitochondrial functions were >1.5-fold downregulated in the spargel mutant fat body (Table 3 and Appendix, section 4.1.). Stefanie Katharina Tiefenböck 32

35 Category # detected (of total #) up down OXPHOS 76 (89) 6 60 TCA 29 (40) 8 12 Ribosomal proteins, protein folding, stabilization 25 (30) 3 18 Protein targeting and proteolysis 20 (30) 3 9 AA metabolism 33 (45) 6 13 Lipids 21 (24) 7 3 DNA and RNA 7 (8) 0 6 Others (Sulfur, nucleotide and cofactor metabolism, transport 41 (47) 4 18 facilitation, cell death and others) Total # 252 (313) Percentage of detected genes Table 3. Categories of mitochondrial functions. Shown is the number of genes that were detected per category. The numbers in brackets indicate the total number of genes annotated per category. Up or down columns show the number of genes that were significantly up- or downregulated >1.5-fold in the srl 1/1 mutant fat body compared their wildtype control (Srl wt/wt ). This included not only genes encoding proteins for mitochondrial function, like complex I-V of the oxidative phosphorylation, but also factors that control the replication and transcription of the mitochondrial genome, such as TFAM, mttfb1, mttfb2 and mtssb. In addition, regulators of mitochondrial translation, like the mitochondria-specific translation elongation factor Tu (EfTuM), and a number of mitochondrial ribosomal proteins, among these mrpl12 and bonsai, were affected. qrt-pcr confirmed the deregulation of these genes (Fig. 11). Stefanie Katharina Tiefenböck 33

36 Figure 11. The effect of Spargel on the mrna expression of selected target genes. The qrt-pcr verification of microarray results of Spargel (A) and selected genes functioning in (A) fatty acid beta-oxidation, (B) mitochondrial oxidative phosphorylation, (C) mitochondrial DNA replication and transcription or (D) mitochondrial translation. The transcript levels were determined by qrt-pcr from RNA isolated from dissected Stefanie Katharina Tiefenböck 34

37 fat bodies (3 biological replicates). As in the microarrays mid-third instar larvae were taken (4d AED for +/+, 5d AED for srl 1 /srl 1 ). Transcript levels were normalized to gammatubulin23c (gtub, CG3157). In all cases, the significance is indicated as compared to the control sample. *** equals P<0.001; ** equals P<0.01; * equals P<0.05. As many aspects of mitochondrial mass and function were downregulated, we further analysed to what extent mitochondrial mass was affected in the spargel mutants and if these mitochondria were functional. As mentioned in the introduction, PGC-1 proteins regulate the expression of nuclear encoded mitochondrial genes by coactivating the NRF-1 and NRF-2 transcription factors as well as the nuclear receptor ERRα. In the fly only Delg, the Drosophila homolog of NRF-2, has been shown to affect mitochondrial gene expression in the larval fat body (Baltzer et al., submitted). Thus, we tested, if analogous to the PGC- 1/NRF-2 interaction, a similar regulatory interaction on gene expression exists in the fly The effect of Spargel in the control of mitochondrial mass and activity and its in vivo interaction with Delg, the mammalian homolog of NRF-2α Spargel and Delg, the Drosophila homolog of mammalian NRF-2a, share many target genes First, we analyzed if Spargel and Delg would have overlapping transcriptional targets. We therefore compared fat body-specific microarray data of spargel or delg single mutants in more detail and focused on genes encoding mitochondrial proteins. Interestingly, we found that of all genes that are downregulated in either the spargel or the delg mutant, about half (88 genes) overlapped (Fig. 12A and Appendix, section 4.1.). This included many OXPHOS and TCA cycle genes. In contrast, 46 genes were downregulated in the spargel, but not in the delg Stefanie Katharina Tiefenböck 35

38 mutant. These genes function in electron transport (complex I), DNA and RNA metabolism and mitochondrial protein synthesis and targeting. In addition, 27 genes were affected in a Delg-specific manner, including genes required for amino acid and fatty acid metabolism. We conclude that Spargel and Delg share many putative target genes, but also affect transcription independently of each other. To verify the microarray data, we again dissected larval fat bodies, and used qrt-pcr to quantify mrna levels of selected genes. Glutamate dehydrogenase (Gdh), involved in amino acid synthesis, was strongly affected in the delg, but not in the spargel mutant. Moreover, we examined two genes that, based on chromatin immunoprecipitation experiments, are direct Delg targets: RFeSP, the Rieske iron-sulfur protein of complex III, and Bellwether (Blw), the ATP synthase subunit alpha of complex V (Baltzer et al., submitted). Both genes were expressed at lower levels in either spargel or delg single mutants. Importantly, spargel delg double mutants did not show a further decrease in RFeSP and Blw mrna levels (Fig. 12B). We conclude that Spargel and Delg have a common role in the expression of many genes encoding mitochondrial proteins, possibly through Spargel-mediated coactivation of Delg. Yet at the same time, either factor is required for expression levels of a subset of these genes independently of the other. Stefanie Katharina Tiefenböck 36

39 Figure 12. Spargel and the NRF-2α homologue Delg share many putative target genes. (A) Comparison of microarray data from srl 1/1 and delg -/- single mutants using mrna of fat bodies dissected from mid-l3 larvae. Shown is the overlap of all nuclear encoded mitochondrial genes that are downregulated >1.5x. Processes that are regulated in a Spargel- or Delg-specific manner, respectively, as well as overlapping gene sets are indicated below. (B) The expression of nuclear encoded mitochondrial proteins (RFeSP, Blw and Gdh) was determined by qrt-pcr from RNA isolated from dissected fat bodies (3 biological replicates). As above, mid-third instar larvae were taken (4d AED for wildtype, 5d AED for spargel, 6d AED for delg, 8d AED for spargel delg). Transcript levels were normalized to gammatubulin23c (gtub, CG3157). In all cases, delg -/- refers to delg 613 /Df(3R)ro 80b. In all cases, the significance is indicated as compared to the control sample Spargel and Delg function in parallel pathways in respect to mitochondrial mass To test whether the reduced expression of genes encoding mitochondrial proteins would result in defective mitochondria we analyzed the mitochondrial mass and activity of the spargel mutants more in detail. In parallel, we tested the in vivo relevance of the results from the microarray comparisons between Spargel and Delg by analyzing the mitochondrial phenotypes in a spargel delg double mutant. As a read-out for mitochondrial mass, we first Stefanie Katharina Tiefenböck 37

40 performed MitoTracker stainings on larval fat bodies of wildtype (+/+), spargel (srl 1/1 ) and delg (delg -/- ) single mutants and the spargel delg double mutants. Similar to the stainings in Chapter , control animals had abundant mitochondrial staining throughout the cytoplasm (Fig. 13A). In spargel mutants, we detected no or only a minimal reduction in staining. In contrast to the spargel single mutants (Baltzer et al., submitted), delg single mutants showed a strong reduction in mitochondrial staining, where residual mitochondria are concentrated around the nucleus. Importantly, spargel delg double mutants had a more severe phenotype compared to the delg single mutant, where only few mitochondria are stained per cell. To complement the MitoTracker staining, we used NAO, which specifically labels the mitochondrial phospholipid cardiolipin. Again, spargel mutant cells showed almost no decrease in staining compared to control, whereas delg mutants had strong defects, which were even exacerbated in the double mutant (Fig. 13B). These data demonstrate that Spargel is not required for mitochondrial mass under normal conditions, but becomes limiting in the absence of Delg suggesting an additive effect of Spargel and Delg in the control of mitochondrial mass. Furthermore, since delg single mutants but not spargel alone showed a reduction in mitochondrial stainings, our findings imply that Delg-specific functions, such as fatty acid and amino acid metabolism (e.g. Gdh), are rate-limiting for mitochondrial mass. The fat body is known to release lipids and amino acids, in particular proline, providing energy sources for other tissues (Baker and Thummel, 2007). Since proline is synthesized from the mitochondrial TCA cycle intermediate 2-oxoglutarate, we propose that such a function could be rate-limiting for mitochondrial mass is the fat body. In contrast, genes that require Spargel for expression, in particular OXPHOS genes, are not determining mitochondrial mass. In agreement, the fat body of wild type larvae does not attract tracheoles for gas exchange (Jarecki et al., 1999), suggesting low rates of mitochondrial respiration. Indeed, compared to other larval tissues like the gut, we detected a low inner-mitochondrial Stefanie Katharina Tiefenböck 38

41 membrane potential and reduced oxygen consumption in the mitochondria of the fat body (Baltzer et al., submitted). Figure 13. Spargel and Delg have additive effects on mitochondrial mass. (A) Mitochondria-specific MitoTracker stainings of larval fat bodies. DAPI is shown as insets. (B) NAO stainings, specific for the mitochondrial phospholipid cardiolipin, in unfixed larval fat bodies. Hoechst is shown as insets. For (A) and (B), bar equals 20µm. Mid-L3 larvae were used, except for the spargel delg double mutant in (A) which was taken at age 5d AED. Kindly provided by Christian Frei. To further test for additive defects, we measured larval growth rates. Whereas spargel or delg single mutants pupated with a 1 or 2-day delay, respectively, double mutants grew very slowly, showing strongly reduced size at 4 days AED, and pupation occurred with a 4-day delay (Fig. 14). This phenotype is similar to mutants lacking the mitochondrial ribosomal protein S15 (Galloni, 2003) or the mitochondrial protein translocator Tim50 (Sugiyama et al., 2007), thus might be caused by additive mitochondrial defects. Alternatively, since Spargel and Delg are required for proper expression of genes involved in translation and ribosome biogenesis (Table 1 and Baltzer et al., submitted), non-mitochondrial functions might be impaired in the double mutant, leading to the apparent growth defects. In either scenario, Stefanie Katharina Tiefenböck 39

42 although Spargel and Delg might function together in the expression of individual genes, these factors act in parallel pathways in respect to mitochondrial mass and larval growth rates. Figure 14. Additive effects on larval growth and development in spargel delg double mutants. (A) Additive effect of Spargel and Delg on larval development. All larvae are at age 4d AED. Bar equals 1mm. (B) Larval volumes of wildtype, spargel or delg single mutants, as well as spargel delg double mutants. Pictures of larvae at the indicated time points were taken and larval volumes were determined by measuring the larval dimensions in Adobe Photoshop and subsequent calculation in Excel using the following formula: 4/3π(Length/2) 2 (Diameter/2) (Colombani et al., 2003). While spargel and delg single mutants shown modest growth defects, they pupate (indicated as P ) with a 1-day or 2-day delay, respectively. The spargel delg double mutants show strongly reduced larval growth rates, and pupate with a 4-day delay. Moreover, spargel delg double mutant pupae are significantly smaller than control or single mutant pupae. Kindly provided by Nicole Egli Oxidative phosphorylation defects in spargel delg double mutant fat bodies To test for the functionality of the mitochondria of the single and double mutants we looked at mitochondrial activity. For this we took several approaches. First, we performed an activity assay for Cytochrome c oxidase (COX), which is part of complex IV in the mitochondrial electron transport chain. Compared to the wildtype, no difference in activity could be observed for the spargel mutant (Fig. 15). As described before (Baltzer et al., submitted) delg Stefanie Katharina Tiefenböck 40

43 mutants showed a higher activity. The spargel, delg double mutant fat bodies had comparable COX activity to the delg single mutant. In contrast to the additive effect on mitochondrial mass, Spargel and Delg show no additive effect on mitochondrial COX activity (Fig. 15). COX activity assay Figure 15. No additive effect of Spargel and Delg on mitochondrial COX activity. Shown is the COX activity assay of mid-3 rd instar larval fat bodies. Inset shows addition of KCN, what inhibits COX activity (kindly provided by Nicole Egli). Another test for OXPHOS activity is the quantification of the mitochondrial respiration. For this, we measured oxygen consumption of dissected, digitonin-permeabilized fat bodies (Fig. 16A). The advantage of this method is that mitochondria can be studied in situ therefore the risk of damaging or losing mitochondria during the isolation procedure is minimized and their physiological environment is preserved (Kuznetsov et al., 2008). Compared to control, spargel mutant fat bodies showed identical respiration upon stimulation of complex I by pyruvate and proline (state 2). Moreover, maximal respiration (state 3; upon addition of ADP), and uncoupled respiration not linked to ATP synthesis (state 4; after the addition the ADP/ATP transporter inhibitor atractyloside) were not changed. When delg single mutants were analyzed, we noted slightly reduced state 2 and state 4 respirations, as well as lower basal oxygen consumption in the presence of the complex IV inhibitor cyanide. However, these defects are compared to the isogenic control of the spargel mutant. When compared to their own control, delg mutants did not show reduced respiration rates (Baltzer et al., submitted) suggesting less abundant yet more active mitochondria. When spargel delg double Stefanie Katharina Tiefenböck 41

44 mutant fat bodies were assayed, we noted increased state 2 and state 4 respiration. This is most likely due to enhanced non-mitochondrial oxygen consumption, since we also detected a respiration increase in the presence of cyanide. Importantly, spargel delg double mutants were not induced to the same extent by the addition of ADP: Whereas the ratio of state 3/state 2 was at least 2.5 for control and single mutant tissues, this ratio was reduced to 2 in the double mutant (Fig. 16B). Since this ratio is indicative of the OXPHOS capacity, these data demonstrate respiration defects in spargel delg double mutant fat bodies. In addition, we noted a decreased respiratory control ratio (RCR; state 3/state 4 ratio), indicative of increased uncoupling. To further analyse the degree of coupling in the mutant mitochondria, we also measured total fat body ATP levels. Confirming the respiration measurements, no difference in ATP content between the spargel mutant compared to wild type could be detected (Fig. 16C). ATP levels in delg mutants were significantly upregulated further supporting the hypothesis of more active mitochondria in this mutant (Baltzer et al., submitted). In the spargel, delg double mutant no difference to wild type could be observed suggesting that the coupled activity of mitochondria in the double mutants is efficient enough to reach wildtype ATP levels. To further look into mitochondrial coupling we determined the expression levels of uncoupling proteins. Quantitative real time PCR showed that, whereas UCP4A is not regulated in any of the mutants, UCP4B and Bmcp are upregulated in the spargel, delg double mutant, but not in either single mutants (Fig. 16D). This is in agreement with the increased uncoupling observed in the double mutant compared to the single mutants. In summary, these data show a common effect of Spargel and Delg on uncoupled respiration, but an independent control of mitochondrial coupled respiration. Given the strong mitochondrial mass reduction in the double mutants (Fig. 13A and 13B), it appears surprising that the tissue respires, even at reduced capacity. However, as described above, OXPHOS is not the predominant function of fat body mitochondria, suggesting that only minimal mitochondrial mass is required. Moreover, a transcription-independent mechanism might compensate for reduced expression Stefanie Katharina Tiefenböck 42

45 of OXPHOS. Alternatively, factors that are rate-limiting for OXPHOS activity might be controlled in a Spargel and Delg-independent manner. Future work is required to test these models. To finish the characterization of mitochondrial function, we quantified the mitochondrial DNA (mtdna), which encodes several factors required for electron transport, and which levels were shown to correlate with OXPHOS activity (Rocher et al., 2008). When normalized to nuclear DNA, we did not detect a change in the spargel single mutant, but increased levels in delg single and the double mutants (Fig. 16E). Importantly, this did not lead to enhanced mtdna transcription, since we detected reduced transcript levels of mitochondrial encoded ND1 (NADH-ubiquinone oxidoreductase chain 1) or COX subunit I (Fig. 16F). Given the general correlation between mtdna replication and transcription in mammalian cells, this appears surprising. There are two possible explanations for this discrepancy: first, the larval fat body is an endoreplicative tissue and cells of the delg single mutant as well as the spargel, delg double mutant show growth defects which is often connected with endoreplication (Edgar and Orr-Weaver, 2001). As the amount of mitochondrial DNA was normalized to genomic DNA levels it is not completely clear whether the mtdna is upregulated in our case or if these cells contain less nuclear DNA. Further analyses are necessary to clarify this. Second, a similar observation has been described recently upon downregulation of mitochondrial transcription factor B2 (Adan et al., 2008). Thus in Drosophila, mtdna transcription can be uncoupled from mtdna levels, at least under mutant conditions and the reduced OXPHOS capacity in the spargel delg double mutant might be caused by defects in mitochondrial transcription. Taken together, we showed that uncoupling and non-mitochondrial respiration is upregulated in the spargel delg double mutant, indicating a common action of the two factors on these processes. On other mitochondrial aspects like coupled respiration, ATP levels, mtdna content, as well as the Stefanie Katharina Tiefenböck 43

46 activity of COX, no additive effects could be observed in the double mutant. In agreement with this, no additive effects on the expression of oxidative phosphorylation genes could be detected in the double mutant (Nicole Egli, Master`s Thesis; data not shown). In summary, these data support that Spargel and Delg function in parallel pathways in vivo. Figure 16. Spargel and Delg function in parallel pathways. (A) Oxygen consumption of digitoninpermeabilized dissected fat bodies. State 2: respiration after stimulation of complex I (addition of pyruvate/proline). State 3: maximal respiration (addition of ADP). State 4: uncoupled respiration (addition of the ADP/ATP transporter inhibitor atractyloside). KCN (complex IV inhibitor) was added to monitor background. Respiration was normalized to total fat body protein (3 biological replicates). (B) Ratios of oxygen consumption from D. The respiratory control ratio (RCR) reflects state 3/4. (C) ATP content was measured from the fat bodies of mid 3 rd instar larvae. (D) The expression of uncoupling proteins UCP4A, UCP4A and Bmcp was analyzed by qrt-pcr on RNA isolated from dissected fat bodies (5 biological replicates, courtesy of Nicole Egli). (E) Mitochondrial DNA (mtdna) content was quantified by qpcr and normalized to nuclear DNA (ndna) levels from dissected fat bodies. Averages and standard deviations were calculated from 6 biological replicates. (F) Mitochondrial-encoded COX (Subunit I) and ND1 were determined by qrt-pcr (3 biological replicates). For (D) and (F): Transcript levels were normalized to gammatubulin23c. We used mid 3 rd instar larvae for all experiments. In all cases, the significance is indicated as compared to the control sample. Stefanie Katharina Tiefenböck 44

47 Discussion Our microarray analysis shows that Drosophila Spargel is predominantly required for the proper expression of genes encoding mitochondrial proteins which is analogous to the function of the mammalian PGC-1 proteins. Importantly, in contrast to its mammalian homologs, Spargel is not a master regulator of mitochondrial biogenesis as we can not detect any obvious changes in mitochondrial mass in the spargel single mutant. Rather, Spargel becomes limiting for these functions in the absence of Delg, a functional and structural NRF- 2α homologue, suggesting an additive effect of Spargel and Delg in the control of mitochondrial mass. Furthermore, we showed that uncoupling and non-mitochondrial respiration is upregulated in the spargel delg double mutant, indicating a common action of the two factors on these processes. On other mitochondrial aspects of mitochondrial activity (respiration efficiency, ATP content and COX activity), however, no additive effects could be observed in the double mutant. This suggests that these two factors act not only on common downstream targets, but also have independent functions in the larval fat body. This clearly differs from the mammalian system, where PGC-1s and NRF-2α function together on expression of nuclear genes encoding mitochondrial proteins. To further investigate these findings interaction studies will be important to reveal whether Spargel and Delg interact directly and whether Spargel has coactivator function. Additionally, as spargel single mutants do affect the expression of genes that control mitochondrial biogenesis (such as mitochondrial transcription and translation factors) electron microscopy experiments should be done to look at mitochondrial morphology and mitochondrial mass in more detail. Also, in analogy to the function of mammalian PGC-1s during certain stress stimuli, mitochondrial function in the spargel mutant could be challenged Stefanie Katharina Tiefenböck 45

48 to test if Spargel is required to modulate adaptations of mitochondrial functions in response to specific stimuli, such as increased activity, cold exposure or reactive oxygen stress. The expression analysis of spargel mutant fat bodies showed that also non-mitochondrial functions were affected. These include major processes involved in the regulation of growth, like gene expression and translation. Because spargel mutant larvae display a defect in developmental size increase, which becomes more dramatic in the spargel delg double mutant, we further analyzed a possible function for Spargel in the control of cellular and organismal growth. Stefanie Katharina Tiefenböck 46

49 2.6. Spargel is required for insulin-signalling mediated growth and controls part of the transcriptional changes induced by insulin-signalling Background There is only little known about the regulation of mitochondria in response to growthpromoting pathways and nutrient availability, so one main interest of my PhD thesis was to elucidate the in vivo regulation of this process. As already mentioned in the introduction, many of the known growth-driving pathways are well conserved in the fly: the insulin/tor pathway (Grewal, 2008), Myc (Eilers and Eisenman, 2008), CyclinD/Cdk4 (Datar et al., 2000; Meyer et al., 2000) or the Hippo tumour-suppressor pathway (Saucedo and Edgar, 2007). Out of these the insulin/tor signalling pathway has been shown to mediate a link between nutrients, cellular metabolism and growth (Britton et al., 2002). Important for this study, recent microarray studies in the fly have shown that starvation, and subsequent reduced insulin-signalling activity, leads to lower expression of genes encoding mitochondrial proteins (Gershman et al., 2007; Teleman et al., 2008). Since the insulin-signalling pathway is well characterized in the fly and many tools are available for genetic and biochemical studies, we chose this pathway to analyze a functional interaction between PGC-1/Spargel, mitochondrial biogenesis and growth control. In flies, the insulin receptor (INR) is activated by the binding of insulin-like peptides (dilps) (Brogiolo et al., 2001) that are released from a cluster of median neurosectrectory cells in the brain, the IPCs (insulin-producing cells), upon specific nutritional cues (Colombani et al., 2003; Ikeya et al., 2002). This stimulates a signalling pathway that includes Chico, the Stefanie Katharina Tiefenböck 47

50 homolog of mammalian Insulin receptor substrate (IRS) as well as the downstream kinases PI3K/Dp110 and PKB/Akt and results in the subsequent inhibition of the forkhead transcription factor dfoxo (Grewal, 2008). Importantly, as multiple proteins have been shown to influence insulin-signalling, this pathway is not linear, but regulates and responds to other signalling pathways (Colombani et al., 2005; Grewal, 2008; Orme et al., 2006; Wang et al., 2005). Accordingly, although dfoxo appears to be the predominant factor to control transcription in respect to insulin signalling, additional transcription factors must exist, since many genes are regulated in a dfoxo-independent manner upon starvation (Teleman et al., 2008) Insulin receptor-signalling requires Spargel to mediate its effects on cellular growth To analyze a possible role for Spargel within the insulin signalling-pathway, we tested if Spargel is required for insulin-signalling to mediate its effect on growth. For this, we ectopically induced cell-autonomous growth by the overexpression of INR (UAS-INR, (Brogiolo et al., 2001)) in random clones that are marked with GFP using the Flip-out/Gal4 system (Tubulin>CD2>Gal4, UAS-GFP (Scott et al., 2004)) and compared the size phenotype in the wildtype to the spargel mutant background. In wildtype fat body cells, the overexpression of UAS-INR led to an enormous overgrowth phenotype that is characterized by an increase in cellular and nuclear areas (Fig. 17A). Interestingly, this is accompanied by a significant reduction in lipid droplet size as revealed by Nile red, a dye specific for lipids. This probably reflects increased demand for energy during cell growth, coupled with enhanced phospholipid usage for membrane synthesis. In contrast to the wildtype background, the effects of INR overexpression on cell growth and lipid droplets are completely abrogated in the spargel mutant background, demonstrating a requirement for Spargel. Stefanie Katharina Tiefenböck 48

51 To genetically characterize Spargel s role in insulin-signalling, we generated double mutants of spargel and chico, the latter being the Drosophila IRS-1 homologue, and the best-studied mutant of the insulin-signalling pathway (Bohni et al., 1999). Chico mutants have significantly reduced adult body weight, thus are similar to spargel mutants, but the effect is stronger. Importantly, we did not observe a further weight reduction in chico spargel double mutants (Fig. 17B), suggesting that Spargel might have an integrate role in the insulinsignalling pathway. Figure 17. Insulin-signalling requires Spargel to mediate its effect on growth. (A) UAS-INR was expressed in random clones using the hs-flp; Tub>CD2>Gal4, UAS-GFP system in wildtype or spargel mutant background. Shown are stainings from larval fat bodies, using Nile red (red) specific for lipid, DAPI (blue) specific for DNA and GFP (green), which marks INR expressing cells. Bar equals 20µm. (B) Weight of adult males, 2 days after eclosion. Genotypes are indicated. *** equals P<0.001; ns: not significant. To further elucidate the dependence on Spargel for insulin-signalling to drive growth, we overexpressed INR (Brogiolo et al., 2001), an activated form of PI3K/Dp110 (Leevers et al., 1996) or myristylated Akt (Stocker et al., 2002) in random clones, and measured cell size using phalloidin, and nuclear size using DAPI (Fig. 18A). As seen in Figure 18B, the increase in cell and nuclear sizes upon INR and Dp110 CAAX expression was significantly suppressed in Stefanie Katharina Tiefenböck 49

52 a spargel mutant background compared to their respective controls. Interestingly, we did not observe a difference in cellular or nuclear areas between wildtype controls and the spargel mutant when overgrowth was induced by myrakt expression. This suggests that Spargel function is required downstream of INR and Dp110, but not Akt. However, we noticed that Akt only induced a relatively mild overgrowth in the fat body cells (Fig. 18B). This is surprising, as the allele used in this experiment was reported to be a potent inducer of overgrowth in the eye (Stocker et al., 2002). In addition, the ectopic activation of myrakt did not lead to increased lipid usage as we could not detect a difference in lipid droplet size (Fig. 18C). To confirm that myrakt function is really activated in this system we performed an antibody staining against dfoxo. Functional myrakt inhibits dfoxo and leads to a relocalization from the nucleus to the cytoplasm. As shown in Figure 18D, we find less nuclear dfoxo in the clones overexpressing myrakt suggesting that Akt protein is induced and functional. One possible explanation for this relatively mild growth induction could be that Akt has only a minor function in the regulation of cell growth in the fat body as compared to the eye. As an alternative, an other Akt isoform than the one used for the generation of this gain-of-function allele could be the important one in the fat body. However, these hypotheses need to be further investigated. Thus, based on the clonal analysis of myrakt overexpression, we can not conclusively determine the genetic interaction of Spargel with Akt. Stefanie Katharina Tiefenböck 50

53 Figure 18. INR and Dp110, but not Akt require Spargel to mediate their effect on growth. (A) Phalloidin staining (red, membranes) of wildtype fat bodies expressing UAS-INR (green) in clones. DAPI marks nuclei (blue). Bar equals 50µm. (B) Quantification of cell area (CA) and nuclear area (NA) of clones expressing UAS- INR, UAS-Dp110 CAAX or UAS-myrAkt in wildtype or srl 1/1 mutant fat bodies. Shown is the fold increase compared to the surrounding control cells (ctrl) not expressing insulin-signalling components. n>25. (C) Clonal expression of UAS-myrAkt in wildtype background. Shown are stainings from larval fat bodies, using Nile red (red) specific for lipid, DAPI (blue) specific for DNA and GFP, which marks myrakt expressing cells. (D) and (E) Antibody staining against dfoxo (red) shows increased translocation of dfoxo out of the nucleus (DAPI, blue) into the cytoplasm in the clones that overexpress myrakt (green) in wildtype (D) or spargel mutant background (E). For (C) to (E): Bar equals 20µm. For (A) and (C) to (E): Clones were induced using the hs-flp; Tub>CD2>Gal4, UAS-GFP system. Stefanie Katharina Tiefenböck 51

54 The observation that Spargel might function downstream of INR and Dp110, but probably not Akt, was further investigated by looking at the interaction of Spargel with dfoxo. Since dfoxo is inhibited by Akt (Junger et al., 2003; Puig et al., 2003), we were interested if Spargel and dfoxo would function together or independently of each other. To analyze this, we performed an α-dfoxo antibody staining of clones overexpressing myrakt in the spargel mutant background. Similar to the wildtype, dfoxo was relocalized from the nucleus to the cytoplasm upon activation of myrakt (Fig. 18E). This shows that the regulation of dfoxo by Akt is not affected in the spargel mutant, suggesting that Spargel is not involved in the control of dfoxo downstream of Akt. Furthermore, a spargel foxo double mutant leads to larval lethality, while both single mutants are viable to adulthood (data not shown). Taken together, these results show that Spargel is required downstream of INR and Dp110 to induce growth, but acts in parallel to Akt and dfoxo Spargel mediates transcription in response to insulin-signalling in parallel to dfoxo As we showed that Spargel is required for insulin-signalling driven growth we hypothesized that this could result from Spargel`s effect on the regulation of gene expression (Chapter 2.4.). Therefore, we asked whether Spargel might function as a transcriptional regulator downstream of insulin-signalling. To test this, we ectopically expressed INR in wildtype and spargel mutant background and used microarray on dissected fat bodies to compare the expression profiles. INR expression was induced by a 30min. heat-shock at 37 C using the above described Flip-out/Gal4 system (Scott et al., 2004). To minimize indirect effects we dissected the fat bodies relatively quickly after INR induction (13 hours after heat-shock). In total, four different genotypes and three biological replicates per genotype were analyzed: the overexpression of INR in the wildtype or spargel mutant background and their respective Stefanie Katharina Tiefenböck 52

55 controls without INR overexpression. The full data set can be accessed at the NCBI database (Edgar et al., 2002) using accession number GSE When INR was overexpressed in a control background, we detected > 90x upregulation of the INR transcript (Log 2 ratio: 6.51, P-value: 1.879x10-6 ; data not shown). This lead to a significant change in expression levels: among 9101 genes that were expressed in the fat body, a total number of 2254 (24.76%) genes were regulated more than 1.5x (Fig. 19A), 1255 genes were upregulated and 999 were downregulated. GO term enrichment analysis showed that processes involved in DNA replication, RNA metabolism and ribosome biogenesis are significantly upregulated (Fig. 19B; see Appendix, section 4.2., for a full list of enriched GO terms). This reflects the increased need for protein synthesis and the increased degree of endoreplication during cellular growth in the fat body. Importantly, we find that many genes encoding for mitochondrial proteins are upregulated by the overexpression of INR. Stefanie Katharina Tiefenböck 53

56 Figure 19. Spargel regulates part of the transcriptional changes in response to insulin-signalling. (A) Shown are the 2254 genes that are up- or downregulated >1.5x in the microarray when INR was overexpressed in the wildtype. Numbers in the white circle indicate how many of these were expressed in a Spargel-dependent manner. (B) GO analysis of all insulin-responsive genes that were >1.5x up- or downregulated. The percentage of genes that are expressed in a Spargel-dependent manner is indicated in the last column. (C) The control of nuclear encoded mitochondrial proteins is regulated in a highly Spargel-dependent manner. (D) qrt-pcr on RNA isolated from dissected fat bodies (3 biological replicates) confirmed the microarray data on the expression of nuclear encoded mitochondrial proteins (mtacp1, Idh and Cyt-c-p). Transcript levels were normalized to Actin5C (CG4027). The significance is compared as indicated. When we looked into the genes encoding mitochondrial proteins more in detail, we found that 33 of 232 detected genes (14.22%) were induced (Fig. 19C; a full list of the expression levels of all genes encoding mitochondrial proteins can be found in Appendix, section 4.1.). In contrast, when INR was expressed in a spargel mutant background, significantly fewer genes Stefanie Katharina Tiefenböck 54

57 were INR-responsive: 8 of 232 detected (3.45%). Thus the majority (75.75%) of INR-induced genes required Spargel, especially genes involved in OXPHOS activity, mitochondrial ribosomal proteins as well as amino acid metabolism (Table 4). We further confirmed these findings by qrt-pcr to detect mrna levels of mtacp1, encoding the mitochondrial Acyl carrier protein 1, Idh, encoding the TCA cycle enzyme Isocitrate dehydrogenase, and Cyt-c-p, encoding Cytochrome c. While INR required Spargel to drive the expression of mtacp1 and Idh, Cyt-c-p did not show such dependence (Fig. 19D). Importantly, the requirement on Spargel for INR to drive gene expression was not limited to mitochondria: when the whole genome was analyzed, 39.84% of INR-responsive genes required Spargel, in particular genes involved in translation, RNA metabolism and transcription (Fig. 19A and 19B; Appendix, section 4.2.). This effect was not due to differences in the expression of INR as INR transcript levels in the spargel mutant background were induced to comparable levels as in the wildtype sample (88.36x upregulated; Log 2 ratio: 6.47, P-value: 1.039x10-6 ). We conclude that Spargel is required to a large extent for the transcriptional control in response to insulin-signalling. Nuclear encoded mitochondrial genes OXPHOS TCA Protein synthesis Protein targeting AA metabolism Lipids Others Total up down up down % of total Table 4. The effect of INR overexpression on the transcription levels of nuclear encoded mitochondrial proteins sorted according to processes. Shown is the number of genes that were significantly up- or downregulated >1.5-fold when UAS-INR was overexpressed in the wildtype or the spargel mutant Stefanie Katharina Tiefenböck 55

58 Since dfoxo is known to partially mediate the transcriptional control in response to insulinsignalling, we compared our data set to published, fat body-specific microarrays, where control or dfoxo mutant larvae were exposed to starvation, reflecting low insulin-signalling (Teleman et al., 2008). Remarkably, we detected only a minimal overlap between Spargeland dfoxo dependent genes, both for genes encoding mitochondrial proteins as well as for the whole genome (data not shown). This together with our genetic data show that Spargel and dfoxo are both essential to control transcription in response to insulin-signalling, yet represent two different output branches. This does however not exclude that Spargel and dfoxo can function together for a subset of genes. Moreover, it was shown that dfoxo controls Spargel expression, at least in S2 cells (Gershman et al., 2007), thus giving a molecular mechanism for such a cooperation The activation of INR leads to increased mitochondrial mass and function The analysis of our microarray data showed that the expression of many genes encoding for mitochondrial proteins is induced upon ectopic INR activation. This goes in line with previous reports that have shown that starvation, and subsequent reduced insulin-signalling activity, leads to lower expression of genes encoding mitochondrial proteins (Gershman et al., 2007; Teleman et al., 2008). As the functional relevance of this observation was not further investigated in these studies, we decided to analyze the effect of INR overexpression in random clones on mitochondrial mass and activity in the larval fat body. Using MitoTracker as a read-out for mitochondrial mass we observed an increase in mitochondrial staining in the clones with induced INR activity compared to the control cells (Fig. 20A). Furthermore, a Cytochrome oxidase C (COX) activity assay showed an increased intensity of the colourreaction in the INR overexpression clones (Fig. 20B), suggesting increased COX activity. Stefanie Katharina Tiefenböck 56

59 Ph.D. Thesis Therefore, the effect on the expression of mitochondrial proteins by activated INR signalling lead to increased mitochondrial biogenesis and activity in the larval fat body. srl1/slr1 B COX +/+ Merge srl1/slr1 MitoTracker +/+ A Figure 20. Overexpression of INR induces mitochondrial mass and activity. (A) MitoTracker (red) stainings of larval fat bodies. Cells that overexpress INR are marked in green, DAPI is shown blue. Bar equals 20µm. (B) Cytochrome C activity assay of fat bodies overexpressing INR in clones. INR expressing cells are marked with an arrow. Bar equals 100µm. For (A) and (B): Clones were induced using the hs-flp; Tub>CD2>Gal4, UASGFP system. Fat bodies from mid-l3 larvae of wildtype and spargel mutants were used. As the transcriptional induction of many INR-responsive genes coding for mitochondrial proteins depended to a large extent on Spargel, we performed the same experiments in a spargel mutant background. Interestingly, spargel could only partially rescue the effect of INR overexpression on mitochondrial mass. In addition, COX activity remained unaffected. Taken together these results suggest that there is at least one parallel pathway to Spargel downstream of INR to induce mitochondrial mass and function (see also Fig. 23). A candidate for this could be the transcription factor dmyc, which was recently shown to be downstream of insulin-signalling in Drosophila (Teleman et al., 2008). In addition, mammalian Myc has Stefanie Katharina Tiefenböck 57

60 been shown to regulate the expression of many nuclear encoded mitochondrial proteins (Li et al., 2005; Kim et al., 2008) Increased Spargel protein levels in response to INR expression Going back to our observation that Spargel is required for INR-induced growth, we investigated a possible mechanism for this interaction. Although PGC-1α and PRC are mostly nuclear proteins (Andersson and Scarpulla, 2001; Puigserver et al., 1998), rat PGC-1α, in response to a stimulus, is known to translocate from the cytoplasm to the nucleus (Wright et al., 2007). To test whether Spargel localization could be affected in response to insulinsignalling, we generated transgenic flies bearing an HA-tagged Spargel on a genomic rescue construct (HA-Srl GR ). When tested by immunofluorescence in the larval fat body, the majority of HA-Spargel was found in the cytoplasm with relatively little staining in the nucleus under physiological conditions (Fig. 21A). When INR was expressed in random clones, we noted a strong increase in HA-Spargel, both in the cytoplasm and to a large extent in the nucleus (Fig. 21C). This result is supported by our microarray study as well as by qrt-pcr, where we detected increased Spargel transcript levels upon INR overexpression (Fig. 21B). These data show that Spargel, at least transcriptionally, is induced by insulin-signalling, leading to elevated Spargel protein, in particular in the nucleus, thus giving a molecular mechanism how Spargel might mediate gene expression in response to insulin-signalling. Stefanie Katharina Tiefenböck 58

61 Figure 21. Insulin-signalling affects Spargel expression levels and protein localization. (A) An HA-tagged Spargel genomic rescue construct (HA-Srl GR ) was used to monitor the subcellular localization of the Spargel protein. Antibody staining against the HA-tag (α-ha; red) shows a predominant cytoplasmic localization. (B) Relative mrna levels of the Spargel transcript were quantified by qrt-pcr on dissected wildtype fat bodies with (+) or without (-) the whole body overexpression of UAS-INR ( INR). (C) UAS-INR was expressed in random clones, marked by GFP as described before. HA-antibody stainings (α-ha; red) and DAPI (blue) reveal an increased localization of HA-Spargel to the nucleus compared to the neighbouring wildtype cells. For A and B, bar equals 20µm Spargel mediates a negative feedback loop on insulin-signalling Since signalling pathways often involve negative feedback loops to dampen signalling activity, we analyzed our microarray data in more detail for genes encoding insulin-signalling components. Indeed, we noted enhanced INR expression in spargel mutants (microarray; confirmed by qrt-pcr: 1.5x change, P value = ), suggesting increased signalling activity. To analyze the insulin-signalling activity in the spargel mutants more in detail, we used the tgph reporter (Britton et al., 2002), which is a fusion protein of the PIP 3 -binding pleckstrin-homology domain (PH) and GFP. In the case of high insulin-signalling activity, Stefanie Katharina Tiefenböck 59

62 PI3K is activated to produce increased levels of PIP 3 in the cell membrane which leads to the recruitment of tgph to the membrane. Indeed, in the spargel mutant fat body we observe enhanced PIP 3 levels at the plasma membrane compared to the wildtype (Fig. 22A). In addition, we monitored the phosphorylation status of Akt, which is known to correlate with Akt activity. Using a phospho-specific Akt antibody we detected increased Akt phosphorylation by immunoblotting (Fig. 22B), further supporting that insulin-signalling activity is increased in the spargel mutant fat body. Thus, Spargel is not only required for insulin-signalling, but also mediates a negative feedback loop, and thus might set a threshold for insulin-signalling to control metabolism. Interestingly, the role of Spargel in the regulation of insulin-signalling is fat body-specific as we do not observe a difference in tgph localization in other tissues, such as salivary glands or gut (data not shown). This suggests a tissue-autonomous effect of Spargel on insulinsignalling. Figure 22. Spargel mediates a negative feed-back loop on insulin-signalling. (A) tgph stainings in the larval fat body, specific for PIP 3 levels in response to PI3K activity (Britton et al., 2002). Bar equals 20µm. (B) Total Akt and phosphorylated Akt (Ser 505 ) levels were detected by western blot and quantified using the Odyssey Detection system. Band intensities were normalized to the respective Tubulin control, and presented as the ratio Stefanie Katharina Tiefenböck 60