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Analysis of the conserved neurotrophic factor MANF in the Drosophila adult brain
Gene Expression Patterns 18 (2015) 8–15
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Gene Expression Patterns j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / g e p
Analysis of the conserved neurotrophic factor MANF in the Drosophila adult brain Vassilis Stratoulias, Tapio I. Heino * Department of Biosciences, University of Helsinki, FI-00014 Helsinki, Finland
A R T I C L E
I N F O
Article history: Received 30 October 2014 Received in revised form 30 March 2015 Accepted 9 April 2015 Available online 24 April 2015 Keywords: MANF Glia Neurons Dopaminergic neurons
A B S T R A C T
Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an evolutionarily conserved neurotrophic factor that supports and protects dopaminergic neurons. The Drosophila MANF (DmMANF) null mutant animals die during early development, and DmMANF is required for the maintenance of dopamine positive neurites. The aim of this study was to investigate the role of DmMANF during later developmental stages. Here we report that DmMANF expression in the adult brain is much wider than in the embryonic and larval stages. It is expressed in both glia and neurons including dopaminergic neurons. Clonal analysis showed that DmMANF is not required cell-autonomously for the differentiation of either glia or dopaminergic neurons. In addition, DmMANF overexpression resulted in no apparent abnormal dopaminergic phenotype while DmMANF silencing in glia resulted in prolonged larval stage. © 2015 Elsevier B.V. All rights reserved.
MANF (mesancephalic astrocyte-derived neurotrophic factor) is an evolutionarily conserved, secreted molecule that shows remarkable amino acid identity between mammals and flies (Apostolou et al., 2008; Lindholm and Saarma, 2010; Palgi et al., 2009; Voutilainen et al., 2009). In mammals, MANF and its paralog CDNF (cerebral dopamine neurotrophic factor) comprise a novel family of neurotrophic factors that is structurally unrelated to classical neurotrophic and growth factors (Lindholm and Saarma, 2010). MANF and CDNF contain a secretion signal, but no pro-sequence, suggesting that they do not need to be enzymatically processed in order to become active. They contain eight spatially conserved cysteine residues which indicate conservation in the secondary structure (Petrova et al., 2003; Shridhar et al., 1996; Voutilainen et al., 2009). MANF and CDNF have been shown to specifically protect dopaminergic neurons both in rat and mouse Parkinson’s disease (PD) models (Lindholm et al., 2008; Voutilainen et al., 2009). Although the mode of action and especially receptors for these molecules are still elusive, recent evidence point toward MANF exerting its neuroprotective effect both intracellularly by inhibiting apoptosis (Hellman et al., 2011) and extracellularly (Voutilainen et al., 2009). Furthermore, MANF has been shown to provide protection against endoplasmic reticulum stress both in in vitro (Apostolou et al., 2008) and in in vivo (Lindahl et al., 2014; Palgi et al., 2012).
Abbreviations: CDNF, cerebral dopamine neurotrophic factor; CNS, central nervous system; MANF, mesencephalic astrocyte-derived neurotrophic factor; PD, Parkinson’s disease; RNAi, RNA interference; TH, tyrosine hydroxylase. * Corresponding author. Department of Biosciences, University of Helsinki, P.O. Box 56 (Viikinkaari 5), FI-00014 Helsinki, Finland. Tel.: +358 504919661. E-mail address: tapio.heino@helsinki.fi (T.I. Heino). http://dx.doi.org/10.1016/j.gep.2015.04.002 1567-133X/© 2015 Elsevier B.V. All rights reserved.
In rodents, MANF and CDNF are expressed widely in both neuronal and non-neuronal tissues (Lindholm et al., 2007, 2008), a common feature for all neurotrophic factors (Sariola, 2001). MANF expression has also been studied in zebrafish where it is expressed widely in the nervous system, as well as in adult organs during development and in adulthood (Chen et al., 2012). Interestingly, morpholino-mediated knockdown of zebrafish MANF did not result in any apparent abnormal phenotype, although it caused a reduction of tyrosine-hydroxylase (TH) positive cells during embryogenesis (Chen et al., 2012). In Drosophila, the single homolog DmMANF is more closely related to MANF than to CDNF (Palgi et al., 2009). DmMANF retains the characteristic spacing of the eight-cysteine residues (Lindholm et al., 2007; Palgi et al., 2009; Petrova et al., 2003; Shridhar et al., 1996), which is critical for the protein folding. During embryonic and larval stages, DmMANF is predominantly expressed in garland cells (Palgi et al., 2012), which are highly endo- and exocytic cells. In addition, during embryonic stages DmMANF is also expressed in salivary glands, Malpighian tubules, fat body, trachea, adult ovaries (Palgi et al., 2009) and pericardial nephrocytes (Stratoulias and Heino, 2015). In the embryonic nervous system, DmMANF is expressed predominantly in glia that surround dopaminergic neurons, but not in neurons (Palgi et al., 2009). DmMANF null mutant animals die during early larval stages and exhibit specific and significant reduction of dopaminergic neurites (Palgi et al., 2009). Interestingly, the lethality of the DmMANF mutants can be rescued with the human MANF, suggesting that the human and the fly MANF proteins are functionally orthologous (Palgi et al., 2009). Recently we showed that concurrent DmMANF knockdown and Dicer-2 overexpression in glia result in the appearance of an unusual macrophage-like cell type in the pupal brain (Stratoulias and Heino, 2015). Surprisingly, this
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Table 1 GAL4 lines with which DmMANF co-localizes in the adult brain.
Glial
Neuronal
a b
Pan-glial Astrocyte-like Cortex Subperineurial Astrocyte-like Perineurial Enseathing Astrocyte-like Pan-neuronal Dopaminergic Neurites
Stock name
DmMANF expression
repo-Gal4 NP1243a NP2222 NP2276 NP3233 NP6293 NP6520b alrm-Gal4 elav-Gal4 TH-Gal4 BP104 and Fas2 antibodies
Yes Yes Yes Yes Yes Yes Yes Yes Partially Yes No
Has secondary expression in Enseathing and Cortex glia. Has secondary expression in Cortex glia.
novel cell type expresses DmMANF and it also appears when either autophagy or immunity is induced in glia (Stratoulias and Heino, 2015). DmMANF expression has not been studied in adult Drosophila except in the ovaries where it is expressed in the follicle and nurse cells (Palgi et al., 2009). Here we focus on DmMANF expression in
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the adult nervous system. We show that in the adult central nervous system (CNS), DmMANF has a much wider distribution compared to its expression during embryonic and larval stages. It co-localizes strongly with glial processes. In addition, we show that contrary to embryos and larvae, in adults DmMANF is localized specifically in dopaminergic cell somas, indicating that DmMANF has a dynamic expression pattern during development. We also investigate whether DmMANF is cell autonomously required for cell differentiation by conducting clonal analysis. Furthermore, we use the UAS/GAL4 system to explore the consequences of silencing of DmMANF function in specific cell types. 1. Results and discussion 1.1. DmMANF is expressed in glial processes It has been shown that during embryonic stages, DmMANF expression is confined to some glial subpopulations, such as glia surrounding dopaminergic neurons, in longitudinal and in channel glia (Palgi et al., 2009). Here we used immunohistochemistry to identify the cell populations where DmMANF is expressed. We found that in the adult brain, DmMANF is expressed in all glial subtypes, as categorized by Awasaki et al. (2008) and Doherty et al. (2009)
Fig. 1. DmMANF co-localizes with glial processes, which radiate in M6 and M7 medulla layers, while they surround dopaminergic neurites. (A–B) DmMANF has a wide expression in the adult brain. DmMANF does not co-localize with the Repo antibody (A), but co-localizes with glial processes (B), as they are marked by repo > UASmCD8::GFP (membrane bound GFP). (C) DmMANF is expressed in glial processes during pupal stages. At 50 hours after puparium formation (APF), DmMANF is expressed in migrating glial (arrow). (D) Red square indicates the area of the brain shown in detail in (E)–(J). (E) DmMANF co-localizes with glial process at the medulla. (F–I) DmMANF positive processes radiate into two layers in the medulla, namely M6 and M7 (auxiliary stainings were performed as in Gao et al., 2008). (J) Interestingly, dopaminergic processes are located between layers M6 and M7 and are surrounded by DmMANF positive layers. White scale bars, 100 μm; orange scale bars, 10 μm.
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Fig. 2. DmMANF is expressed in neurons. (A) DmMANF is found in the cytoplasm of both neurons and glia. (B–D) In neurons, DmMANF does not co-localize with processes. (F–G) DmMANF co-localizes with dopaminergic cell somas (see also Fig. 3). White scale bars, 100 μm; orange scale bars, 10 μm.
(Table 1), thus having a considerably broader expression pattern compared to embryonic stages. At the subcellular level, DmMANF is localized in all glial processes during the adult (Fig. 1B), the mid(Fig. 1C) and late pupal stages. Interestingly, contrary to Drosophila, in rodents MANF is not expressed in glia cells (Lindholm et al., 2008; Shen et al., 2012). The Drosophila optic lobe is very well characterized both anatomically and developmentally (Chotard and Salecker, 2007; Fischbach and Hiesinger, 2008); therefore, we looked in more detail the DmMANF positive glial projections within this area (Fig. 1D). We found that DmMANF positive glial projections radiate specifically in strata M6 and M7 of the medulla neuropil (Fig. 1E–H). Interestingly, dopaminergic neurons exist in the Drosophila optic lobe and project their processes to two layers, M1 and M7. M1 is immediately proximal to the DmMANF positive cell bodies, while M7 is between the strata that DmMANF positive processes radiate (Fig. 1J). The close proximity of the radiated DmMANF positive glial processes and the dopaminergic neurites in the medulla suggest a possible role of DmMANF in the dopaminergic system.
1.2. DmMANF is expressed in neuronal cell somas, including dopaminergic cell somas In rodent brains, MANF is relatively widely expressed in neurons including dopaminergic neurons (Lindholm et al., 2008; Wang et al., 2014), but not in glia (Lindholm et al., 2008; Shen et al., 2012). In zebrafish, MANF is also expressed in neurons and to a smaller extent also in glia (Chen et al., 2012). In Drosophila embryos, however, DmMANF expression has not been detected in any neuronal populations (Palgi et al., 2009). We used co-localization studies to investigate whether DmMANF is expressed in adult neurons. We found that in the adult brain, DmMANF does not co-localize with either of the neuronal axonal markers Fas2 or BP104 (Fig. 2C–D), but it partially co-localizes with the pan-neuronal marker Elav in the neuronal cell somas (Fig. 2E–F). Some of these DmMANF positive neurons stain also with the dopaminergic marker TH, suggesting that DmMANF is expressed in dopaminergic neurons (Figs. 2F–G, 3B). Further analysis revealed that DmMANF is expressed in the cell somas of all seven major dopaminergic clusters of the brain (Fig. 3A).
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Fig. 3. DmMANF is expressed in all seven major dopaminergic clusters of the central brain. (A) The dopaminergic neurons of the Drosophila adult brain are grouped into bilateral symmetric clusters, as presented in the schematic drawing. DmMANF is expressed in all seven major dopaminergic clusters. (B, D) DmMANF co-localizes with THpositive cell somas (B), but not with their processes, even when DmMANF is overexpressed under the TH promoter (D, arrows). (C–C’) Both DmMANF and TH immunoreactivity increase significantly in TH positive cell somas, when DmMANF is overexpressed under a TH promoter. Note that the confocal laser power in (B) and (C) are the same, while in (C’) laser power is greatly decreased. Asterisks in C–C’ indicate TH positive neuron that it is not recognized by α-TH antibody, but expresses TH-Gal4 (see Friggi-Grelin et al., 2003). White scale bars, 25 μm; orange scale bars, 10 μm.
In addition, and contrary to DmMANF expression in glia, DmMANF was not detected in the processes of dopaminergic neurons, not even when DmMANF was overexpressed under the TH-Gal4 driver (Fig. 3D, arrows). Interestingly, DmMANF overexpression in the dopaminergic neurons resulted in increased TH immunoreactivity (Fig. 3B–C’). These results show that DmMANF has a dynamic expression pattern during development of the Drosophila nervous system. 1.3. DmMANF is not required in a cell autonomous manner for the survival and differentiation of either glia or dopaminergic neurons DmMANFΔ96 null mutants are larval lethal (Palgi et al., 2009). Therefore, in order to study the role of DmMANF in the adult brain, we employed techniques such as RNA interference (RNAi) and clonal analysis. To create DmMANFΔ96 homozygous mutant clones in the optic lobe, we used the FRT/FLP technique (Xu and Rubin, 1993). These clones expressed the pan-glial marker Repo (Fig. 4A). We also employed the MARCM technique (Wu and Luo, 2007) to see if DmMANF is needed for the survival and differentiation of dopaminergic neurons. The DmMANFΔ96 homozygous mutant clones expressed the dopaminergic marker TH (Fig. 4B). Based on these results we conclude that DmMANF is not required in a cell autonomous fashion for the survival and differentiation of either glia or dopaminergic neurons. 1.4. DmMANF overexpression does not affect the dopaminergic system MANF has been shown to protect and even rescue the midbrain dopaminergic neurons in vertebrate models of Parkinson’s disease (Lindholm et al., 2008; Voutilainen et al., 2009). In addition, it has been shown to be overexpressed under ischemic stress
(Glembotski et al., 2012; Tadimalla et al., 2008) as well as in activated microglia (Shen et al., 2012). In DmMANF null mutant flies, the dopaminergic neurites are diminished (Palgi et al., 2009). We wanted to explore whether DmMANF overexpression has an effect on the dopaminergic system in adults. In all cases, animals developed normally and lived for at least 30 days. We quantified the TH positive neurons in elav-Gal4; UAS-DmMANF, repo-Gal4; UASDmMANF and TH-Gal4; UAS-DmMANF 5 day old adult brains and we found no statistical significant changes in the number of dopaminergic neuron somas (Fig. 5). This supports our previous observations that DmMANF overexpression does not have apparent phenotypic effects in the nervous system or elsewhere (Palgi et al., 2012 and Lindström et al., in preparation). 1.5. DmMANF and the adult dopaminergic system Next, we knocked down DmMANF mRNA by employing the RNAi technique coupled with the UAS/Gal4 system (Dietzl et al., 2007). Ubiquitous knockdown of DmMANF using either tub-Gal4 or daGal4 resulted into early larval lethality which phenocopies the DmMANFΔ96 null mutant phenotype. To examine if reduced levels of DmMANF in glia has an effect on the adult dopaminergic neurons, we used repo-Gal4 to knockdown DmMANF in all glia. In this case, both male and female animals developed into adults. We also quantified the TH positive neurons in repo-Gal4; DmMANFRNAi 5 day old adult brains and as in the case of the UAS-DmMANF overexpression studies, no statistically significant changes were observed in the number of dopaminergic neuron somas (Fig. 5). Next, we enhanced the efficacy of the RNAi effect by overexpressing Dicer-2, an approach that is commonly used in Drosophila (Dietzl et al., 2007). However, this led to pupal lethality even when the flies were reared at 18 °C (Stratoulias and Heino, 2015). At 26 °C, the repo-Gal4;
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Fig. 4. DmMANF is not needed cell autonomously for specification of either glial or dopaminergic neurons. (A) DmMANF mutant clones that do not express DmMANF differentiate into glial cells which express the pan-glial marker Repo (non-GFP positive cells, circles). (B) DmMANF mutant clones that do not express DmMANF (GFP positive cells, arrows) are specified to become dopaminergic neurons and express TH. Genotypes: (A) hsFLP; lama-Gal4 FRT DmMANFΔ96/FRT UAS-nGFP. (B) MARCM clones: hsFLP; UASmCD8::GFP, TH > FRT DmMANFΔ96/FRT82B tub-Gal80. White scale bars, 10 μm.
UAS-DmMANFRNAi UAS-Dicer-2 animals died as 3rd instar larvae but remained in this stage for 8 days compared to 2 days of the control (repo-Gal4; UAS-Dicer-2) and wild type larvae (n = 20). In addition, the repo-Gal4; UAS-DmMANFRNAi UAS-Dicer-2 larvae showed a locomotion phenotype with reduced peristaltic contraction frequency and circular path trajectories, a phenotype that has been attributed to loss of normal postural control due to interneuron inactivation (Iyengar et al., 2011) (Appendix: Supplementary Movie S1). Furthermore, these animals stayed at the bottom of the tube, they did not climb and they were immobile compared to control and wild-type third-instar larvae (Supplementary Movie S1). Next, we investigated whether DmMANF knockdown in neurons has an effect on the dopaminergic system. First, we used the panneuronal driver elav-Gal4 to knock down DmMANF in all neurons. Quantification of the TH-positive neurons in adult brains showed no significant differences compared to the wild type brains (Fig. 5). Since DmMANF is expressed in dopaminergic neurons (see earlier), we used TH-Gal4 to knock down DmMANF specifically in these neurons. Again, quantification of the TH-positive neurons showed no significant differences compared to the wild type brains (Fig. 5). This result implies that either DmMANF is not required in a cellautonomous manner to dopaminergic neurons or that TH promoter is not strong enough to silence DmMANF in these neurons. However, we noticed sporadic absence of dopamine neuron clusters in TH-Gal4;
UAS-DmMANFRNAi and elav-Gal4; UAS-DmMANF brains (Fig. 6). It has earlier been shown that TH-specific expression of Dicer-2 in dopaminergic neurons has a significant effect on motor behavior in Drosophila and possibly also on dopaminergic neuron survival (White et al., 2010). In accordance with this, we noticed dopaminergic phenotypes in TH-Gal4; UAS-Dicer-2 adult brains (not shown) and we were therefore reluctant to use this enhancement of RNAi in the dopamine neurons. In summary, further studies are required in order to solve the role of DmMANF in the adult dopaminergic system.
2. Experimental procedures 2.1. Fly strains Flies were kept and grown under standard conditions. w1118 flies were regarded as wild type. For the statistical analysis (Figs 5 and 6), Canton-S flies were used as wild type. We used the following stocks from Bloomington Drosophila Stock Centre (BDSC): UASnGFP (4775), UAS-mCD8::GFP (5137), hsFLP (8862), Kr/CyO; D/Tb Sb (7199), FRT82B Gal80 (5135) and UAS-DmMANFRNAi [from Vienna Drosophila RNAi Centre (VDRC) v12835]. The following stocks are the glial subtype drivers obtained from the Drosophila Genomics Resource Centre (DGRC): NP1243 (12835), NP2222 (112830), NP2276
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Fig. 5. DmMANF downregulation and overexpression does not produce statistical significant changes in the number of TH positive neurons during development. (A–E) DmMANF knockdown in either glia (repo>), or neurons (elav>) or specifically in dopaminergic neurons (TH>), does not produce a significant change in the number of TH positive cell somas in PAL, PPL1, PPL2ab, PPM 1/2 and PPM 3 dopaminergic clusters. The same is true for DmMANF overexpression in glia, neurons and dopaminergic neurons. (F) Scheme of the central brain indicating the location of the dopaminergic clusters that were counted. All animals were 5 day old adults. n numbers were as follows: Canton-S (n = 12), repo > MANFRNAi (n = 10), elav > MANFRNAi (n = 12), TH > MANFRNAi (n = 12), repo > UAS-DmMANF (n = 10), elav > UAS-DmMANF (n = 12) and TH > UAS-DmMANF (n = 8). In all cases, p > 0.05 compared with wild type Canton-S flies.
(112853), NP3233 (113173), NP6293 (105188), NP6520 (105240) and alrm-Gal4 [(gift from M. R. Freeman) (Doherty et al., 2009)]. In addition, these stocks were also used: da-Gal4 (Wodarz et al., 1995), elav-Gal4 (BDSC, 8760), lama-Gal4 [(gift from I. Salecker) (Chotard
et al., 2005)], TH-Gal4 [(gift from S. Birman) (Friggi-Grelin et al., 2003)], repo-Gal4 (gift from V. J. Auld), tub-Gal4 (O’Donnell et al., 1994), UAS-Dicer-2 (gift from S. Thor), UAS-DmMANF135 (Palgi et al., 2009) and DmMANFΔ96 (Palgi et al., 2009).
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Fig. 6. DmMANF levels can sporadically influence dopaminergic neuron development. (A) Dopaminergic neuron clusters always appear as mirror image. (B, D) Wild type brains, where the PAL (B), the PPM1/2 (B) and the PPM3 (D) clusters are indicated by geometrical shapes with solid line. (C, E) In some cases, downregulation (C) or upregulation (E) of DmMANF expression resulted into disappearance of TH positive clusters, as indicated by geometrical shapes with dashed line. White scale bars, 100 μm.
2.2. Generation of somatic clones Somatic clones were performed as described in Perrimon (1998). To generate somatic clones, animals of the following genotype were used: hsFLP; lama-Gal4 FRT82B DmMANFΔ96/FRT82B UAS-nGFP. 2.3. Generation of MARCM clones MARCM was performed as described in Wu and Luo (2007). To generate DmMANF null MARCM clones in dopaminergic neurons, +/Y; UASmCD8::GFP; TH-Gal4 FRT82B DmMANFΔ96/Tb Sb males were crossed to hsFLP; +; FRT82B tub-Gal80 females. 2.4. Immunohistochemistry Immunohistochemistry was performed as described in Wu and Luo (2006). The following antibodies were used: rabbit antiDmMANF [1:1000 (Palgi et al., 2009)], mouse anti-TH (Diasorin, 1:25). In addition, the following antibodies from Developmental Studies Hybridoma Bank were also used: mouse anti-Bruchpilot (nc82, 1:10), mouse anti-chaoptin (24B10, 1:100), mouse anti-Discs large (Dlg, 1:10), Rat anti-Elav (1:20), FasII (1D4, 1:100), FasIII (7G10, 1:25), Neuroglian (BP104, 1:10) and mouse anti-Repo (1:10). 2.5. Confocal microscopy and image processing Confocal microscopy was performed using a Leica TCS SP5 confocal microscope. Images were acquired at a resolution of 512 × 512 or 1024 × 1024 and with a line average of 3. Z-projections were created using ImageJ and images were processed using Adobe Photoshop. 2.6. Quantification of TH positive neurons Whole-mount 5 day old adult brains were labeled with antiTH (Diasorin, 1:25). Images were acquired at a resolution of 512 × 512 and step size 2 μm. TH labeled cells were counted manually through each z-stack using the Leica Application Suite Advanced Fluorescence (LAS AF) version 2.6.0 software. Numbers of cells were recorded per hemisphere for PAL, PPL1, PPL2ab, PPM1/2 and PPM3
cluster. The mean number of cells per cluster and per hemisphere was calculated. Statistical analysis was performed with independent samples t-test. Acknowledgements Most of the antibodies were provided by the Development Studies Hybridoma Bank, and the fly strains were from the Bloomington Drosophila Stock Centre, the Vienna Drosophila RNAi Centre and the Drosophila Genomics Resource Centre. The authors would like to thank Iris Salecker for providing the lama-Gal4 fly stock and Arja Ikävalko for her assistance with crosses and stock maintenance. This work was supported in parts by the Academy of Finland, Marie Curie Early Stage Training, the Finnish Cultural Foundation, University of Helsinki funds and the Ella and Georg Ehrnrooth Foundation. Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.gep.2015.04.002. References Apostolou, A., Shen, Y., Liang, Y., Luo, J., Fang, S., 2008. Armet, a UPR-upregulated protein, inhibits cell proliferation and ER stress-induced cell death. Exp. Cell Res. 314, 2454–2467. Awasaki, T., Lai, S., Ito, K., Lee, T., 2008. Organization and postembryonic development of glial cells in the adult central brain of Drosophila. J. Neurosci. 28, 13742–13753. Chen, Y., Sundvik, M., Rozov, S., Priyadarshini, M., Panula, P., 2012. MANF regulates dopaminergic neuron development in larval zebrafish. Dev. Biol. 370, 237–249. Chotard, C., Salecker, I., 2007. Glial cell development and function in the Drosophila visual system. Neuron Glia Biol. 3, 17–25. Chotard, C., Leung, W., Salecker, I., 2005. glial cells missing and gcm2 cell autonomously regulate both glial and neuronal development in the visual system of Drosophila. Neuron 48, 237–251. Dietzl, G., Chen, D., Schnorrer, F., Su, K., Barinova, Y., Fellner, M., et al., 2007. A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature 448, 151–156. Doherty, J., Logan, M.A., Taşdemir, Ö.E., Freeman, M.R., 2009. Ensheathing glia function as phagocytes in the adult Drosophila brain. J. Neurosci. 29, 4768–4781. Fischbach, K., Hiesinger, P.R., 2008. Optic Lobe Development. Brain Development in Drosophila Melanogaster. Springer, pp. 115–136. Friggi-Grelin, F., Coulom, H., Meller, M., Gomez, D., Hirsh, J., Birman, S., 2003. Targeted gene expression in Drosophila dopaminergic cells using regulatory sequences from tyrosine hydroxylase. J. Neurobiol. 54, 618–627.
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Petrova, P.S., Raibekas, A., Pevsner, J., Vigo, N., Anafi, M., Moore, M.K., et al., 2003. MANF. A new mesencephalic, astrocyte-derived neurotrophic factor with selectivity for dopaminergic neurons. J. Mol. Neurosci. 20, 173–187. Sariola, H., 2001. The neurotrophic factors in non-neuronal tissues. Cell. Mol. Life Sci. 58, 1061–1066. Shen, Y., Sun, A., Wang, Y., Cha, D., Wang, H., Wang, F., et al., 2012. Upregulation of mesencephalic astrocyte-derived neurotrophic factor in glial cells is associated with ischemia-induced glial activation. J. Neuroinflammation 9, 254. Shridhar, V., Rivard, S., Shridhar, R., Mullins, C., Bostick, L., Sakr, W., et al., 1996. A gene from human chromosomal band 3p21.1 encodes a highly conserved arginine-rich protein and is mutated in renal cell carcinomas. Oncogene 12, 1931–1939. Stratoulias, V., Heino, T.I., 2015. MANF silencing, immunity induction or autophagy trigger an unusual cell type in metamorphosing Drosophila brain. Cell. Mol. Life Sci. 72, 1989–2004. Tadimalla, A., Belmont, P.J., Thuerauf, D.J., Glassy, M.S., Martindale, J.J., Gude, N., et al., 2008. Mesencephalic astrocyte-derived neurotrophic factor is an ischemiainducible secreted endoplasmic reticulum stress response protein in the heart. Circ. Res. 103, 1249–1258. Voutilainen, M.H., Bäck, S., Pörsti, E., Toppinen, L., Lindgren, L., Lindholm, P., et al., 2009. Mesencephalic astrocyte-derived neurotrophic factor is neurorestorative in rat model of Parkinson’s disease. J. Neurosci. 29, 9651–9659. Wang, H., Ke, Z., Alimov, A., Xu, M., Frank, J.A., Fang, S., et al., 2014. Spatiotemporal expression of MANF in the developing rat brain. PLoS ONE 9, e90433. White, K.E., Humphrey, D.M., Hirth, F., 2010. The dopaminergic system in the aging brain of Drosophila. Front. Neurosci. 4, 205. Wodarz, A., Hinz, U., Engelbert, M., Knust, E., 1995. Expression of crumbs confers apical character on plasma membrane domains of ectodermal epithelia of Drosophila. Cell 82, 67–76. Wu, J.S., Luo, L., 2006. A protocol for dissecting Drosophila melanogaster brains for live imaging or immunostaining. Nat. Protoc. 1, 2110–2115. Wu, J.S., Luo, L., 2007. A protocol for mosaic analysis with a repressible cell marker (MARCM) in Drosophila. Nat. Protoc. 1, 2583–2589. Xu, T., Rubin, G.M., 1993. Analysis of genetic mosaics in developing and adult Drosophila tissues. Development 117, 1223–1237.
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Gene Expression Patterns j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / g e p
Analysis of the conserved neurotrophic factor MANF in the Drosophila adult brain Vassilis Stratoulias, Tapio I. Heino * Department of Biosciences, University of Helsinki, FI-00014 Helsinki, Finland
A R T I C L E
I N F O
Article history: Received 30 October 2014 Received in revised form 30 March 2015 Accepted 9 April 2015 Available online 24 April 2015 Keywords: MANF Glia Neurons Dopaminergic neurons
A B S T R A C T
Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an evolutionarily conserved neurotrophic factor that supports and protects dopaminergic neurons. The Drosophila MANF (DmMANF) null mutant animals die during early development, and DmMANF is required for the maintenance of dopamine positive neurites. The aim of this study was to investigate the role of DmMANF during later developmental stages. Here we report that DmMANF expression in the adult brain is much wider than in the embryonic and larval stages. It is expressed in both glia and neurons including dopaminergic neurons. Clonal analysis showed that DmMANF is not required cell-autonomously for the differentiation of either glia or dopaminergic neurons. In addition, DmMANF overexpression resulted in no apparent abnormal dopaminergic phenotype while DmMANF silencing in glia resulted in prolonged larval stage. © 2015 Elsevier B.V. All rights reserved.
MANF (mesancephalic astrocyte-derived neurotrophic factor) is an evolutionarily conserved, secreted molecule that shows remarkable amino acid identity between mammals and flies (Apostolou et al., 2008; Lindholm and Saarma, 2010; Palgi et al., 2009; Voutilainen et al., 2009). In mammals, MANF and its paralog CDNF (cerebral dopamine neurotrophic factor) comprise a novel family of neurotrophic factors that is structurally unrelated to classical neurotrophic and growth factors (Lindholm and Saarma, 2010). MANF and CDNF contain a secretion signal, but no pro-sequence, suggesting that they do not need to be enzymatically processed in order to become active. They contain eight spatially conserved cysteine residues which indicate conservation in the secondary structure (Petrova et al., 2003; Shridhar et al., 1996; Voutilainen et al., 2009). MANF and CDNF have been shown to specifically protect dopaminergic neurons both in rat and mouse Parkinson’s disease (PD) models (Lindholm et al., 2008; Voutilainen et al., 2009). Although the mode of action and especially receptors for these molecules are still elusive, recent evidence point toward MANF exerting its neuroprotective effect both intracellularly by inhibiting apoptosis (Hellman et al., 2011) and extracellularly (Voutilainen et al., 2009). Furthermore, MANF has been shown to provide protection against endoplasmic reticulum stress both in in vitro (Apostolou et al., 2008) and in in vivo (Lindahl et al., 2014; Palgi et al., 2012).
Abbreviations: CDNF, cerebral dopamine neurotrophic factor; CNS, central nervous system; MANF, mesencephalic astrocyte-derived neurotrophic factor; PD, Parkinson’s disease; RNAi, RNA interference; TH, tyrosine hydroxylase. * Corresponding author. Department of Biosciences, University of Helsinki, P.O. Box 56 (Viikinkaari 5), FI-00014 Helsinki, Finland. Tel.: +358 504919661. E-mail address: tapio.heino@helsinki.fi (T.I. Heino). http://dx.doi.org/10.1016/j.gep.2015.04.002 1567-133X/© 2015 Elsevier B.V. All rights reserved.
In rodents, MANF and CDNF are expressed widely in both neuronal and non-neuronal tissues (Lindholm et al., 2007, 2008), a common feature for all neurotrophic factors (Sariola, 2001). MANF expression has also been studied in zebrafish where it is expressed widely in the nervous system, as well as in adult organs during development and in adulthood (Chen et al., 2012). Interestingly, morpholino-mediated knockdown of zebrafish MANF did not result in any apparent abnormal phenotype, although it caused a reduction of tyrosine-hydroxylase (TH) positive cells during embryogenesis (Chen et al., 2012). In Drosophila, the single homolog DmMANF is more closely related to MANF than to CDNF (Palgi et al., 2009). DmMANF retains the characteristic spacing of the eight-cysteine residues (Lindholm et al., 2007; Palgi et al., 2009; Petrova et al., 2003; Shridhar et al., 1996), which is critical for the protein folding. During embryonic and larval stages, DmMANF is predominantly expressed in garland cells (Palgi et al., 2012), which are highly endo- and exocytic cells. In addition, during embryonic stages DmMANF is also expressed in salivary glands, Malpighian tubules, fat body, trachea, adult ovaries (Palgi et al., 2009) and pericardial nephrocytes (Stratoulias and Heino, 2015). In the embryonic nervous system, DmMANF is expressed predominantly in glia that surround dopaminergic neurons, but not in neurons (Palgi et al., 2009). DmMANF null mutant animals die during early larval stages and exhibit specific and significant reduction of dopaminergic neurites (Palgi et al., 2009). Interestingly, the lethality of the DmMANF mutants can be rescued with the human MANF, suggesting that the human and the fly MANF proteins are functionally orthologous (Palgi et al., 2009). Recently we showed that concurrent DmMANF knockdown and Dicer-2 overexpression in glia result in the appearance of an unusual macrophage-like cell type in the pupal brain (Stratoulias and Heino, 2015). Surprisingly, this
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Table 1 GAL4 lines with which DmMANF co-localizes in the adult brain.
Glial
Neuronal
a b
Pan-glial Astrocyte-like Cortex Subperineurial Astrocyte-like Perineurial Enseathing Astrocyte-like Pan-neuronal Dopaminergic Neurites
Stock name
DmMANF expression
repo-Gal4 NP1243a NP2222 NP2276 NP3233 NP6293 NP6520b alrm-Gal4 elav-Gal4 TH-Gal4 BP104 and Fas2 antibodies
Yes Yes Yes Yes Yes Yes Yes Yes Partially Yes No
Has secondary expression in Enseathing and Cortex glia. Has secondary expression in Cortex glia.
novel cell type expresses DmMANF and it also appears when either autophagy or immunity is induced in glia (Stratoulias and Heino, 2015). DmMANF expression has not been studied in adult Drosophila except in the ovaries where it is expressed in the follicle and nurse cells (Palgi et al., 2009). Here we focus on DmMANF expression in
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the adult nervous system. We show that in the adult central nervous system (CNS), DmMANF has a much wider distribution compared to its expression during embryonic and larval stages. It co-localizes strongly with glial processes. In addition, we show that contrary to embryos and larvae, in adults DmMANF is localized specifically in dopaminergic cell somas, indicating that DmMANF has a dynamic expression pattern during development. We also investigate whether DmMANF is cell autonomously required for cell differentiation by conducting clonal analysis. Furthermore, we use the UAS/GAL4 system to explore the consequences of silencing of DmMANF function in specific cell types. 1. Results and discussion 1.1. DmMANF is expressed in glial processes It has been shown that during embryonic stages, DmMANF expression is confined to some glial subpopulations, such as glia surrounding dopaminergic neurons, in longitudinal and in channel glia (Palgi et al., 2009). Here we used immunohistochemistry to identify the cell populations where DmMANF is expressed. We found that in the adult brain, DmMANF is expressed in all glial subtypes, as categorized by Awasaki et al. (2008) and Doherty et al. (2009)
Fig. 1. DmMANF co-localizes with glial processes, which radiate in M6 and M7 medulla layers, while they surround dopaminergic neurites. (A–B) DmMANF has a wide expression in the adult brain. DmMANF does not co-localize with the Repo antibody (A), but co-localizes with glial processes (B), as they are marked by repo > UASmCD8::GFP (membrane bound GFP). (C) DmMANF is expressed in glial processes during pupal stages. At 50 hours after puparium formation (APF), DmMANF is expressed in migrating glial (arrow). (D) Red square indicates the area of the brain shown in detail in (E)–(J). (E) DmMANF co-localizes with glial process at the medulla. (F–I) DmMANF positive processes radiate into two layers in the medulla, namely M6 and M7 (auxiliary stainings were performed as in Gao et al., 2008). (J) Interestingly, dopaminergic processes are located between layers M6 and M7 and are surrounded by DmMANF positive layers. White scale bars, 100 μm; orange scale bars, 10 μm.
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Fig. 2. DmMANF is expressed in neurons. (A) DmMANF is found in the cytoplasm of both neurons and glia. (B–D) In neurons, DmMANF does not co-localize with processes. (F–G) DmMANF co-localizes with dopaminergic cell somas (see also Fig. 3). White scale bars, 100 μm; orange scale bars, 10 μm.
(Table 1), thus having a considerably broader expression pattern compared to embryonic stages. At the subcellular level, DmMANF is localized in all glial processes during the adult (Fig. 1B), the mid(Fig. 1C) and late pupal stages. Interestingly, contrary to Drosophila, in rodents MANF is not expressed in glia cells (Lindholm et al., 2008; Shen et al., 2012). The Drosophila optic lobe is very well characterized both anatomically and developmentally (Chotard and Salecker, 2007; Fischbach and Hiesinger, 2008); therefore, we looked in more detail the DmMANF positive glial projections within this area (Fig. 1D). We found that DmMANF positive glial projections radiate specifically in strata M6 and M7 of the medulla neuropil (Fig. 1E–H). Interestingly, dopaminergic neurons exist in the Drosophila optic lobe and project their processes to two layers, M1 and M7. M1 is immediately proximal to the DmMANF positive cell bodies, while M7 is between the strata that DmMANF positive processes radiate (Fig. 1J). The close proximity of the radiated DmMANF positive glial processes and the dopaminergic neurites in the medulla suggest a possible role of DmMANF in the dopaminergic system.
1.2. DmMANF is expressed in neuronal cell somas, including dopaminergic cell somas In rodent brains, MANF is relatively widely expressed in neurons including dopaminergic neurons (Lindholm et al., 2008; Wang et al., 2014), but not in glia (Lindholm et al., 2008; Shen et al., 2012). In zebrafish, MANF is also expressed in neurons and to a smaller extent also in glia (Chen et al., 2012). In Drosophila embryos, however, DmMANF expression has not been detected in any neuronal populations (Palgi et al., 2009). We used co-localization studies to investigate whether DmMANF is expressed in adult neurons. We found that in the adult brain, DmMANF does not co-localize with either of the neuronal axonal markers Fas2 or BP104 (Fig. 2C–D), but it partially co-localizes with the pan-neuronal marker Elav in the neuronal cell somas (Fig. 2E–F). Some of these DmMANF positive neurons stain also with the dopaminergic marker TH, suggesting that DmMANF is expressed in dopaminergic neurons (Figs. 2F–G, 3B). Further analysis revealed that DmMANF is expressed in the cell somas of all seven major dopaminergic clusters of the brain (Fig. 3A).
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Fig. 3. DmMANF is expressed in all seven major dopaminergic clusters of the central brain. (A) The dopaminergic neurons of the Drosophila adult brain are grouped into bilateral symmetric clusters, as presented in the schematic drawing. DmMANF is expressed in all seven major dopaminergic clusters. (B, D) DmMANF co-localizes with THpositive cell somas (B), but not with their processes, even when DmMANF is overexpressed under the TH promoter (D, arrows). (C–C’) Both DmMANF and TH immunoreactivity increase significantly in TH positive cell somas, when DmMANF is overexpressed under a TH promoter. Note that the confocal laser power in (B) and (C) are the same, while in (C’) laser power is greatly decreased. Asterisks in C–C’ indicate TH positive neuron that it is not recognized by α-TH antibody, but expresses TH-Gal4 (see Friggi-Grelin et al., 2003). White scale bars, 25 μm; orange scale bars, 10 μm.
In addition, and contrary to DmMANF expression in glia, DmMANF was not detected in the processes of dopaminergic neurons, not even when DmMANF was overexpressed under the TH-Gal4 driver (Fig. 3D, arrows). Interestingly, DmMANF overexpression in the dopaminergic neurons resulted in increased TH immunoreactivity (Fig. 3B–C’). These results show that DmMANF has a dynamic expression pattern during development of the Drosophila nervous system. 1.3. DmMANF is not required in a cell autonomous manner for the survival and differentiation of either glia or dopaminergic neurons DmMANFΔ96 null mutants are larval lethal (Palgi et al., 2009). Therefore, in order to study the role of DmMANF in the adult brain, we employed techniques such as RNA interference (RNAi) and clonal analysis. To create DmMANFΔ96 homozygous mutant clones in the optic lobe, we used the FRT/FLP technique (Xu and Rubin, 1993). These clones expressed the pan-glial marker Repo (Fig. 4A). We also employed the MARCM technique (Wu and Luo, 2007) to see if DmMANF is needed for the survival and differentiation of dopaminergic neurons. The DmMANFΔ96 homozygous mutant clones expressed the dopaminergic marker TH (Fig. 4B). Based on these results we conclude that DmMANF is not required in a cell autonomous fashion for the survival and differentiation of either glia or dopaminergic neurons. 1.4. DmMANF overexpression does not affect the dopaminergic system MANF has been shown to protect and even rescue the midbrain dopaminergic neurons in vertebrate models of Parkinson’s disease (Lindholm et al., 2008; Voutilainen et al., 2009). In addition, it has been shown to be overexpressed under ischemic stress
(Glembotski et al., 2012; Tadimalla et al., 2008) as well as in activated microglia (Shen et al., 2012). In DmMANF null mutant flies, the dopaminergic neurites are diminished (Palgi et al., 2009). We wanted to explore whether DmMANF overexpression has an effect on the dopaminergic system in adults. In all cases, animals developed normally and lived for at least 30 days. We quantified the TH positive neurons in elav-Gal4; UAS-DmMANF, repo-Gal4; UASDmMANF and TH-Gal4; UAS-DmMANF 5 day old adult brains and we found no statistical significant changes in the number of dopaminergic neuron somas (Fig. 5). This supports our previous observations that DmMANF overexpression does not have apparent phenotypic effects in the nervous system or elsewhere (Palgi et al., 2012 and Lindström et al., in preparation). 1.5. DmMANF and the adult dopaminergic system Next, we knocked down DmMANF mRNA by employing the RNAi technique coupled with the UAS/Gal4 system (Dietzl et al., 2007). Ubiquitous knockdown of DmMANF using either tub-Gal4 or daGal4 resulted into early larval lethality which phenocopies the DmMANFΔ96 null mutant phenotype. To examine if reduced levels of DmMANF in glia has an effect on the adult dopaminergic neurons, we used repo-Gal4 to knockdown DmMANF in all glia. In this case, both male and female animals developed into adults. We also quantified the TH positive neurons in repo-Gal4; DmMANFRNAi 5 day old adult brains and as in the case of the UAS-DmMANF overexpression studies, no statistically significant changes were observed in the number of dopaminergic neuron somas (Fig. 5). Next, we enhanced the efficacy of the RNAi effect by overexpressing Dicer-2, an approach that is commonly used in Drosophila (Dietzl et al., 2007). However, this led to pupal lethality even when the flies were reared at 18 °C (Stratoulias and Heino, 2015). At 26 °C, the repo-Gal4;
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Fig. 4. DmMANF is not needed cell autonomously for specification of either glial or dopaminergic neurons. (A) DmMANF mutant clones that do not express DmMANF differentiate into glial cells which express the pan-glial marker Repo (non-GFP positive cells, circles). (B) DmMANF mutant clones that do not express DmMANF (GFP positive cells, arrows) are specified to become dopaminergic neurons and express TH. Genotypes: (A) hsFLP; lama-Gal4 FRT DmMANFΔ96/FRT UAS-nGFP. (B) MARCM clones: hsFLP; UASmCD8::GFP, TH > FRT DmMANFΔ96/FRT82B tub-Gal80. White scale bars, 10 μm.
UAS-DmMANFRNAi UAS-Dicer-2 animals died as 3rd instar larvae but remained in this stage for 8 days compared to 2 days of the control (repo-Gal4; UAS-Dicer-2) and wild type larvae (n = 20). In addition, the repo-Gal4; UAS-DmMANFRNAi UAS-Dicer-2 larvae showed a locomotion phenotype with reduced peristaltic contraction frequency and circular path trajectories, a phenotype that has been attributed to loss of normal postural control due to interneuron inactivation (Iyengar et al., 2011) (Appendix: Supplementary Movie S1). Furthermore, these animals stayed at the bottom of the tube, they did not climb and they were immobile compared to control and wild-type third-instar larvae (Supplementary Movie S1). Next, we investigated whether DmMANF knockdown in neurons has an effect on the dopaminergic system. First, we used the panneuronal driver elav-Gal4 to knock down DmMANF in all neurons. Quantification of the TH-positive neurons in adult brains showed no significant differences compared to the wild type brains (Fig. 5). Since DmMANF is expressed in dopaminergic neurons (see earlier), we used TH-Gal4 to knock down DmMANF specifically in these neurons. Again, quantification of the TH-positive neurons showed no significant differences compared to the wild type brains (Fig. 5). This result implies that either DmMANF is not required in a cellautonomous manner to dopaminergic neurons or that TH promoter is not strong enough to silence DmMANF in these neurons. However, we noticed sporadic absence of dopamine neuron clusters in TH-Gal4;
UAS-DmMANFRNAi and elav-Gal4; UAS-DmMANF brains (Fig. 6). It has earlier been shown that TH-specific expression of Dicer-2 in dopaminergic neurons has a significant effect on motor behavior in Drosophila and possibly also on dopaminergic neuron survival (White et al., 2010). In accordance with this, we noticed dopaminergic phenotypes in TH-Gal4; UAS-Dicer-2 adult brains (not shown) and we were therefore reluctant to use this enhancement of RNAi in the dopamine neurons. In summary, further studies are required in order to solve the role of DmMANF in the adult dopaminergic system.
2. Experimental procedures 2.1. Fly strains Flies were kept and grown under standard conditions. w1118 flies were regarded as wild type. For the statistical analysis (Figs 5 and 6), Canton-S flies were used as wild type. We used the following stocks from Bloomington Drosophila Stock Centre (BDSC): UASnGFP (4775), UAS-mCD8::GFP (5137), hsFLP (8862), Kr/CyO; D/Tb Sb (7199), FRT82B Gal80 (5135) and UAS-DmMANFRNAi [from Vienna Drosophila RNAi Centre (VDRC) v12835]. The following stocks are the glial subtype drivers obtained from the Drosophila Genomics Resource Centre (DGRC): NP1243 (12835), NP2222 (112830), NP2276
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Fig. 5. DmMANF downregulation and overexpression does not produce statistical significant changes in the number of TH positive neurons during development. (A–E) DmMANF knockdown in either glia (repo>), or neurons (elav>) or specifically in dopaminergic neurons (TH>), does not produce a significant change in the number of TH positive cell somas in PAL, PPL1, PPL2ab, PPM 1/2 and PPM 3 dopaminergic clusters. The same is true for DmMANF overexpression in glia, neurons and dopaminergic neurons. (F) Scheme of the central brain indicating the location of the dopaminergic clusters that were counted. All animals were 5 day old adults. n numbers were as follows: Canton-S (n = 12), repo > MANFRNAi (n = 10), elav > MANFRNAi (n = 12), TH > MANFRNAi (n = 12), repo > UAS-DmMANF (n = 10), elav > UAS-DmMANF (n = 12) and TH > UAS-DmMANF (n = 8). In all cases, p > 0.05 compared with wild type Canton-S flies.
(112853), NP3233 (113173), NP6293 (105188), NP6520 (105240) and alrm-Gal4 [(gift from M. R. Freeman) (Doherty et al., 2009)]. In addition, these stocks were also used: da-Gal4 (Wodarz et al., 1995), elav-Gal4 (BDSC, 8760), lama-Gal4 [(gift from I. Salecker) (Chotard
et al., 2005)], TH-Gal4 [(gift from S. Birman) (Friggi-Grelin et al., 2003)], repo-Gal4 (gift from V. J. Auld), tub-Gal4 (O’Donnell et al., 1994), UAS-Dicer-2 (gift from S. Thor), UAS-DmMANF135 (Palgi et al., 2009) and DmMANFΔ96 (Palgi et al., 2009).
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Fig. 6. DmMANF levels can sporadically influence dopaminergic neuron development. (A) Dopaminergic neuron clusters always appear as mirror image. (B, D) Wild type brains, where the PAL (B), the PPM1/2 (B) and the PPM3 (D) clusters are indicated by geometrical shapes with solid line. (C, E) In some cases, downregulation (C) or upregulation (E) of DmMANF expression resulted into disappearance of TH positive clusters, as indicated by geometrical shapes with dashed line. White scale bars, 100 μm.
2.2. Generation of somatic clones Somatic clones were performed as described in Perrimon (1998). To generate somatic clones, animals of the following genotype were used: hsFLP; lama-Gal4 FRT82B DmMANFΔ96/FRT82B UAS-nGFP. 2.3. Generation of MARCM clones MARCM was performed as described in Wu and Luo (2007). To generate DmMANF null MARCM clones in dopaminergic neurons, +/Y; UASmCD8::GFP; TH-Gal4 FRT82B DmMANFΔ96/Tb Sb males were crossed to hsFLP; +; FRT82B tub-Gal80 females. 2.4. Immunohistochemistry Immunohistochemistry was performed as described in Wu and Luo (2006). The following antibodies were used: rabbit antiDmMANF [1:1000 (Palgi et al., 2009)], mouse anti-TH (Diasorin, 1:25). In addition, the following antibodies from Developmental Studies Hybridoma Bank were also used: mouse anti-Bruchpilot (nc82, 1:10), mouse anti-chaoptin (24B10, 1:100), mouse anti-Discs large (Dlg, 1:10), Rat anti-Elav (1:20), FasII (1D4, 1:100), FasIII (7G10, 1:25), Neuroglian (BP104, 1:10) and mouse anti-Repo (1:10). 2.5. Confocal microscopy and image processing Confocal microscopy was performed using a Leica TCS SP5 confocal microscope. Images were acquired at a resolution of 512 × 512 or 1024 × 1024 and with a line average of 3. Z-projections were created using ImageJ and images were processed using Adobe Photoshop. 2.6. Quantification of TH positive neurons Whole-mount 5 day old adult brains were labeled with antiTH (Diasorin, 1:25). Images were acquired at a resolution of 512 × 512 and step size 2 μm. TH labeled cells were counted manually through each z-stack using the Leica Application Suite Advanced Fluorescence (LAS AF) version 2.6.0 software. Numbers of cells were recorded per hemisphere for PAL, PPL1, PPL2ab, PPM1/2 and PPM3
cluster. The mean number of cells per cluster and per hemisphere was calculated. Statistical analysis was performed with independent samples t-test. Acknowledgements Most of the antibodies were provided by the Development Studies Hybridoma Bank, and the fly strains were from the Bloomington Drosophila Stock Centre, the Vienna Drosophila RNAi Centre and the Drosophila Genomics Resource Centre. The authors would like to thank Iris Salecker for providing the lama-Gal4 fly stock and Arja Ikävalko for her assistance with crosses and stock maintenance. This work was supported in parts by the Academy of Finland, Marie Curie Early Stage Training, the Finnish Cultural Foundation, University of Helsinki funds and the Ella and Georg Ehrnrooth Foundation. Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.gep.2015.04.002. References Apostolou, A., Shen, Y., Liang, Y., Luo, J., Fang, S., 2008. Armet, a UPR-upregulated protein, inhibits cell proliferation and ER stress-induced cell death. Exp. Cell Res. 314, 2454–2467. Awasaki, T., Lai, S., Ito, K., Lee, T., 2008. Organization and postembryonic development of glial cells in the adult central brain of Drosophila. J. Neurosci. 28, 13742–13753. Chen, Y., Sundvik, M., Rozov, S., Priyadarshini, M., Panula, P., 2012. MANF regulates dopaminergic neuron development in larval zebrafish. Dev. Biol. 370, 237–249. Chotard, C., Salecker, I., 2007. Glial cell development and function in the Drosophila visual system. Neuron Glia Biol. 3, 17–25. Chotard, C., Leung, W., Salecker, I., 2005. glial cells missing and gcm2 cell autonomously regulate both glial and neuronal development in the visual system of Drosophila. Neuron 48, 237–251. Dietzl, G., Chen, D., Schnorrer, F., Su, K., Barinova, Y., Fellner, M., et al., 2007. A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature 448, 151–156. Doherty, J., Logan, M.A., Taşdemir, Ö.E., Freeman, M.R., 2009. Ensheathing glia function as phagocytes in the adult Drosophila brain. J. Neurosci. 29, 4768–4781. Fischbach, K., Hiesinger, P.R., 2008. Optic Lobe Development. Brain Development in Drosophila Melanogaster. Springer, pp. 115–136. Friggi-Grelin, F., Coulom, H., Meller, M., Gomez, D., Hirsh, J., Birman, S., 2003. Targeted gene expression in Drosophila dopaminergic cells using regulatory sequences from tyrosine hydroxylase. J. Neurobiol. 54, 618–627.
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