i MECHANISMS UNDERLYING DENDRITE PRUNING OF DROSOPHILA DENDRITIC ARBORIZATION NEURONS GU YING (B. Sci., Sichuan University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2009 ii To my parents and grandparents iii Acknowledgement I am heartily thankful to my supervisor, Dr. Fengwei Yu, whose encouragement, guidance and support enabled me to explore any possibilities in my research work. His great enthusiasm in science is always stimulating to me. I am also grateful to Assoc. Prof. Boon Chuan Low for his willingness to co-supervise me from the very beginning of my study and support on my work. I also would like to thank my graduate committee members, Prof. William Chia, Dr. Suresh Jeuthasan, Dr. Sudipto Roy and Dr. Yih Cherng Liou for their support and advice. I gratefully thank all people in Yu’s group for providing a motivating, enthusiastic and critical atmosphere for my work, especially Daniel Kirilly for his willingness to share his bright thoughts with me and assistance in various ways. Many thanks also go to Dr. Hongyan Wang and her group for their discussion, in particular to Hongyan, Nick Bogard and Wei Leong Chew for their constructive comments on this thesis. I owe my deepest gratitude to Dr. Arash Bashirullah (UW-Madison), Prof. Alex Kolodkin (HHMI, Johns Hopkins University) and a broader fly community for their generosity in sharing reagents and flies. My gratitude also goes to the supporting staffs and my friends at Temasek Life Sciences Laboratory for their sincere help. And lastly, my parents and grandparents, for their love. iv Table of Contents Acknowledgement . iii Table of Contents . iv Summary . vii List of Publications viii List of Tables . ix List of Figures . x List of Abbreviations xii Chapter One: Literature Review . 1.1 Introduction . 1.2 Neuronal pruning 1.2.1 Developmental pruning in vertebrates 1.2.1.1 Trophic-factor dependent axon pruning 1.2.1.2 Axon guidance molecules in developmental axon pruning 1.2.1.3 Developmental dendrite pruning in vertebrates 1.2.2 Two systems in Drosophila to study developmentally occurring neuronal remodeling 1.3 Mechanisms in regulating neuronal pruning in Drosophila . 1.3.1 Transcriptional regulation of neuronal pruning during metamorphosis . 1.3.2 Ubiquitin-proteasome System . 14 1.3.3 Caspase and neuronal pruning 15 1.3.4 IKK-related kinase IK2, cytoskeleton and dendrite severing . 16 1.4 Aim of this study . 16 Chapter Two: Materials and Methods . 18 2.1 Fly Strains . 18 2.2 Genetic mapping . 19 2.3 Microscopy and image acquisition and quantification . 19 2.4 MARCM labeling . 20 2.5 Fluorescence in situ Hybridization . 21 2.5.1 Primer design for DNA template 21 2.5.2 In vitro transcription of the probe . 21 2.5.3 In situ hybridization 22 2.6 Immunohistochemistry . 23 2.7 DNA manipulations 23 2.7.1 Escherichia. coli culture and transformation 23 v 2.7.2 Molecular cloning . 24 2.7.3 DNA sequencing . 24 2.7.4 Genomic DNA extraction . 25 2.7.5 mical promoter-lacZ reporter plasmid constructs . 26 2.7.6 Mical domain deletion plasmid constructs . 27 2.7.7 Mical single domain plasmid constructs . 28 2.8 Preparation of whole animal lysates, SDS-PAGE and western blot . 29 Chapter Three: Results 31 3.1 Dendrite remodeling of ddaC neurons during metamorphosis . 31 3.2 Forward genetic screen for novel players in ddaC dendrite pruning 32 3.3 Mical regulates dendrite pruning of dendritic arborization neurons in Drosophila 35 3.3.1 Mical is affected in l(3)15256 with strong dendrite severing defects . 35 3.3.2 Mical promotes dendrite severing of ddaCs . 36 3.3.3 Cell-autonomous function of Mical for dendrite severing 38 3.3.4 Time-course analysis of EcR and Mical in dendrite severing 41 3.3.5 Temporal expression pattern of EcR-B1 and Mical . 43 3.3.6 Identification of ecdysone response element(s) in the mical regulatory region ……………………………………………………………………………48 3.3.7 Mical does not affect EcR-B1 expression . 54 3.3.8 Mical is a crucial factor downstream of EcR-B1 to promote dendrite severing . 56 3.3.9 Mical and EcR-B1 are insufficient at early stage to cause premature pruning 58 3.3.10 Functional analysis of Mical domains in dendrite severing 60 3.3.11 Cytoskeleton rearrangement in mical15256 during dendrite pruning 64 3.4 Dronc and Mical regulate different cellular responses to EcR-B1 . 68 3.4.1 Dronc is not required for dendrite severing of ddaCs . 68 3.4.2 Mical is not required for cell death of apoptotic neurons . 73 3.5 Plexin/Semaphorin pathway is not required for ddaC dendrite pruning . 73 3.6 Candidate gene analysis in dendrite pruning 77 Chapter Four: Discussions 80 4.1 Questions about the developmentally regulated neuronal remodeling in Drosophila 80 4.2 Developmental regulation of Mical expression during dendrite pruning . 81 4.3 Mical or Dronc in regulating dendrite severing 84 4.4 How Mical regulates dendrite pruning and future directions . 85 Chapter Five: Conclusions 91 Bibliography . 93 Appendix i . 104 vi Appendix ii 105  vii Summary The capability of neurons to remodel existing neuronal projections and connections confers great flexibility in response to activity-dependent processes, developmental regulated alterations, neuronal diseases and post-injury recoveries. Although a wide range of events lead to neuronal remodeling, the underlying mechanisms remain elusive. Among various types of neuronal remodeling, selective removal of dendrite branches, so called dendrite pruning, of Drosophila dendritic arborization (da) neurons occurs during metamorphosis, a developmental process that transforms a ‘worm-like’ larva into an adult fruit fly. To understand the mechanisms that regulate dendrite pruning of these peripheral neurons, a forward genetic screen was carried out and identified Mical (Molecule interacting with CasL) as a novel factor that promotes severing of dendrites at the initial stage of dendrite pruning. Further studies suggest that destabilization of cytoskeleton molecules, such as microtubules and actins, is suppressed in remodeling da neurons devoid of Mical. Mical functions in da neuron pruning downstream of the steroid nuclear hormone receptor complex EcR-B1/Ultraspirical during the larval-pupal transition; whereas Dronc (Drosophila Nedd2-like caspase) mediates cell-death of apoptotic da neurons and clearance of dendrite debris. viii List of Publications Kirilly D*, Gu Y*, Huang Y, Wu Z, Bashirullah A, Low BC, Kolodkin AL, Wang H, Yu F (2009) A novel pathway composed of Sox14 and Mical governs severing of dendrites during pruning. Nature Neuroscience 12: 1497‐1505 (*as co-first author) ix List of Tables Table 1. Summary in mapping results of mutant lines with dendrite pruning defects… 34 x List of Figures Figure 1. Drosophila dendritic arborization (da) neuron as a model system to study dendrite remodeling during metamorphosis. 10  Figure 2. Dendrite pruning defects of 15 EMS-induced mutant lines. 33  Figure 3. Mical is required for dendrite severing of ddaCs. . 37  Figure 4. Cell-autonomous function of Mical for dendrite severing. . 40  Figure 5. Time-course analysis of ddaC pruning behavior in wt, EcR-B1DN, usp RNAi and mical mutant 42  Figure 6. Time-course analysis of class I neuron ddaD/E pruning behavior in wt, EcRB1DN and mical mutant 44  Figure 7. Mical expression in ddaCs is dependent on EcR-B1/Usp. 47  Figure 8. Ecdysone-responsive elements in the mical regulatory region. 51  Figure 9. Activation of ecdysone-responsive elements of the mical regulatory region in MB γ neurons. . 53  Figure 10. EcR-B1 expression is not affected by Mical. 55  Figure 11. Mical promotes dendrite severing downstream of EcR-B1. . 57  Figure 12. Overexpression of EcR-B1 or Mical by itself is not sufficient to cause precocious pruning. . 59  Figure 13. Mical domain analysis with deletion constructs in mical mutant ddaCs . 61  Figure 14. Mical domain analysis with deletion constructs in wt ddaCs 63  Figure 15. Mical domain analysis with single domain constructs in wt ddaCs. . 65  Figure 16. Cytoskeleton markers in mical 15256. . 66  Figure 17. Dronc is not required for dendrite severing with Mical. . 71  Figure 18. Dronc is required for apoptotic neuron cell death but not for remodeling neuron pruning. . 72  Figure 19. Mical is not required for cell death of apoptotic neuron during metamorphosis. . 74  90 provides explanations for our observation that overexpressing the full-length Mical alone cannot lead to premature dendrite severing, because the excessive Mical may not in its active form or the activation mechanism may also be temporally regulated at the onset of metamorphosis. Worthy of mention here is that Mical is conserved from flies to mammals. The human genome encodes three Mical family members (H-Mical-1/2/3) (Terman et al., 2002) and mRNA of rodent Mical-1/2/3 has been detected to be widely expressed in the embryonic, postnatal and adult nervous system (Pasterkamp et al., 2006). As mentioned earlier in mouse models, Plexins and Semaphorins have been proposed to mediate axon pruning of the hippocampal neuron mossy fiber. However, the mechanism in their study on how axon pruning is achieved is not fully understood, whether it is through axon terminal retraction or local degeneration. Thus, it is very interesting to know whether mammalian Micals are also involved in such developmental pruning processes and whether Mical expression is regulated by hormone or local signals. Besides the study of mammalian Mical function in neurons, Mical has also been implicated to interact with several GTP-bound form of Rab proteins via its C terminus in non-neuronal cell cultures, such as Rab13 in mediating the endocytic recycling of tight junction components (Terai et al., 2006) or Rab1 which participates in regulating vesicle transportation of ER to Golgi (Weide et al., 2003; Fischer et al., 2005). It is not known how much analogy is shared across different systems. However, all studies favor a model that Mical may function as a scaffold protein that assembles intracellular molecules to transduce signals on the cytoskeleton. 91 Chapter Five: Conclusions Our study in understanding the mechanisms underlying dendrite pruning of Drosophila dendritic arborization neuron C (ddaC) started from the time-lapse analysis of dendrite pruning process of this neuron. During metamorphosis, complex dendritic arbors of ddaC undergo successive stages of regressive events, including dendrite severing, fragmentation and clearance. These events involve dynamic reorganization of cytoskeleton molecules such as microtubules and actins. To achieve the temporal regulation of dendrite pruning, ecdysone signaling upregulates its nuclear receptor EcR-B1 expression in response to the ecdysone pulse at the onset of metamorphosis. The activation of ecdysone-bound EcR-B1 receptor together with its co-receptor Usp elicits a cascade of transcriptional activation events that lead to the upregulation of Mical, a newly identified target of ecdysone signaling from this study in promoting dendrite severing of remodeling da neurons (including class IV ddaCs and class I ddaDs and ddaEs). Mical is expressed in all da neurons with a low level of expression at early larval stages and a high level upon puparium formation. Although Mical does not seem to be involved in dendrite morphogenesis of larval ddaCs, removal of Mical in ddaCs dramatically suppresses re-distribution of cytoskeleton components at the initial stage of dendrite severing. Mical encodes a large cytosolic protein with multiple domains, including FM, CH, LIM, Proline-Rich region, Coiled-Coil domain and PDZ-binding motif. 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Development 130: 2603-2610 104 Appendix i LB liquid medium (pH7.5) 1% tryptone 0.5% yeast extract 0.5% NaCl hybridization buffer 50% formamide 5XSSC 100μg/mL salmon sperm DNA 50μG/mL heprin 0.1%Tween20 2x SDS loading buffer 100mM Tris (pH 6.8) 4% SDS 0.2% bromophenol blue 20% glycerol 200mM DTT 1x SDS running buffer Tris Base 3g glycine 14.4g SDS 1g top up to 1L with ddH2O 1x transfer buffer Tris Base 3g glycine 14.4g Methanol 200 mL top up to 1L with ddH2O 105 Appendix ii Gal4 lines Expression pattern in Drosophila nervous system Gal4109(2)80 all da neurons (Gao et al., 1999) Gal42-21 high level in class I da neurons and low level in class IV da neurons (Grueber et al., 2003a) class IV da neurons (Ainsley et al., 2003) for 3rd chromosome insertion line; expressed high in class IV da neurons and low in class III da neurons for 2nd chromosome insertion line (observation from this study) ppk-Gal4 elavC155-Gal4 all neurons (Schuster et al., 1996) 201Y-Gal4 primarily in mushroom body γ neurons (Tettamanti et al.,1997) [...]... For instance, the transcriptional regulation mediated by ecdysone signaling has been shown to control the axon /dendrite pruning events in fly CNS and PNS; and some components of the protein degradation machinery, the ubiquitin-proteasome system (UPS), were identified to be involved in axon pruning of γ neurons as well as dendrite pruning of da neurons 1.3 Mechanisms in regulating neuronal pruning in Drosophila. .. microtubule-severing activity in mediating dendrite pruning of ddaC neuron (Lee et al., 2009) However, it is not known whether overexpression of this microtubule severing factor is sufficient to induce dendrite pruning at an earlier time point 1.4 Aim of this study Regarding mechanisms that regulate developmental dendrite pruning in Drosophila, although molecules ranging from transcription factors to protein destruction... ubiquitination pathway, such as the E1 ubiquitin activation enzyme Uba1, the E2 ubiquitin conjugating enzyme UbcD1, and subunits of the 19S regulatory particle of proteasome, Mov34 and Rpn6, have been identified as regulators of either axon or dendrite pruning or both Interestingly, UbcD1 is specifically required for dendrite pruning instead of axon pruning, although in the MB neurons UbcD1 in indeed... utilized, indicating a common upstream transcriptional control for both dendrite pruning (Kuo et al., 2005) and axon pruning (Lee et al., 2000) Interfering with ecdysone signaling by the neuronal overexpression of the dominant negative form of EcR-B1 (EcR-B1DN) can abolish the dendrite pruning process, by preventing the destabilization of dendritic microtubules and the severing of dendrites Loss of usp... points However, difficulties in identifying, recording and manipulating such a dynamic process in vivo impede us from understanding the underlying mechanisms of developmental pruning 3 1.2.1 Developmental pruning in vertebrates 1.2.1.1 Trophic-factor dependent axon pruning Recent studies of the developmental axon pruning in mammals have shed light on the mechanism underlying this process Neuronal culture... 2006) and from Lee and colleagues’ study, IK2 indeed affects the integrity of Tubulin-GFP and Actin-GFP signal during dendrite pruning, it is possible that besides inhibition of DIAP1, IK2 has a much broader role in dendrite pruning, including cytoskeleton re-organization In addition to the potential impact of IK2 on cytoskeleton, a molecule named Katanin p60-like 1, isolated from a recent RNAi screen... study of the superior cervical ganglion neruons suggested that dendritic morphology is constantly changing in adult mice (Purves et al., 1986), previous investigations of dendrite remodeling have been mainly focused on activity-dependent changes of dendritic spines instead of large-scale dendrite pruning during animal development However, recent studies revealed that the dendritic differentiation of 6... possibilities still remain For instance, processing of molecules by the UPS or caspase could be required for elimination of inhibitors that repress dendrite pruning Such molecules can be transcriptional repressors or cytoskeletonbinding proteins that maintain cytoskeleton integrity at larval stages Alternatively, the processing of molecules could also lead to the activation of signaling molecules that are... the link that is still missing includes how caspases execute the dendrite severing process and the subsequent phagocytosis and how the DIAP1 16 activity is locally regulated in dendrites while keeping the soma alive And it is also interesting to know the reason why the reliance of caspase on dendrite pruning versus axon pruning differs 1.3.4 IKK-related kinase IK2, cytoskeleton and dendrite severing... neuronal remodeling with higher resolution In Drosophila, a single neuron can be labeled (Lee and Luo, 2001) and its morphological changes during development can be traced in real time in vivo imaging Moreover, easy genetic manipulation in the fruit fly confers a great advantage in dissecting the mechanisms of neuronal remodeling One fascinating phenomenon of Drosophila life cycle is that the insect goes . players in ddaC dendrite pruning 32 3.3 Mical regulates dendrite pruning of dendritic arborization neurons in Drosophila 35 3.3.1 Mical is affected in l(3)15256 with strong dendrite severing defects. occurring neuronal remodeling 6 1.3 Mechanisms in regulating neuronal pruning in Drosophila 9 1.3.1 Transcriptional regulation of neuronal pruning during metamorphosis 9 1.3.2 Ubiquitin-proteasome. relevance of the intrinsic machinery and extrinsic machinery of neuronal remodeling since bathing in an environment created by neighboring cells, neurons are constantly exposed to a variety of extracellular