INTERACTIONS BETWEEN EARLY GERM CELLS AND INNER GERMARIAL SHEATH CELLS IN DROSPHILA OVARY LIU MING (M.S Peking Union Medical College, B.S Northeast Normal University, China) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY TEMASEK LIFESCIENCES LABORATORY & DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2009 ACKNOWLEDGEMENTS This thesis work was conducted in Dr Yu CAI’s lab, Germ Cell Development Group, Temasek LifeSciences Laboratory, National University of Singapore, Singapore I thank my three supervisors: Dr Yu CAI, A/Prof Titmeng LIM, and Prof William CHIA for their accepting me as a graduate student Their insightful suggestions, helpful guidance and critical comments have been invaluable in shaping this work and thesis to its present form I also thank all the members of the Yu CAI’s lab, Dr Toshie KAI’s Lab and Bill CHIA’s lab Thanks to Zhou-hua LI, Li-wei WANG, Xin NIE, Liheng TAO, Ai-khim LIM, Jun-wei PEK, Kai-chen CHANG, Sarada, and Phing-chian CHIA for their help and suggestions on my work I am grateful to the members of my thesis committee, Drs Xiaohang YANG, Toshie KAI and Cherng-yih LIOU for their suggestions and comments Many thanks to a lot of other people, especially those at the Bloomington Drosophila center, the many people from the fly community, and facilities in TLL and DBS, who have generously given me reagents at various stages during this work Many thanks go to a lot of my friends in and out of the labs Lastly, I thank my family, especially my parents, elder brothers and sisters-in-law, for all their encouragements and supports i TABLE OF CONTENTS ACKNOWLEDGEMENTS i LIST OF FIGURES AND TABLES vii ABBREVIATIONS x SUMMARY xiii Chapter I Introduction .1 1.1 GSCs and the GSC Niche in Drosophila Ovary 1.1.1 GSCs and Their Intrinsic Factors 1.1.1.1 Drosophila GSCs 1.1.1.2 Intrinsic Factors for GSC Maintenance 1.1.1.3 Intrinsic Factors for CB Differentiation 1.1.2 GSC Microenvironment (Niche) and the Extrinsic Factors in Drosophila Ovary 11 1.2 GSC Niche Activities in Drosophila Ovary .12 1.2.1 Molecules Involved in GSC Niche Activities 12 1.2.2 Signaling Pathways Involved in GSC Niche Activities .13 1.3 Roles of EGFR Signaling in GSC Maintenance and Differentiation (Part I) 16 1.4 Glypicans and Their Functions in Drosophila (Par I) 22 1.4.1 Glypicans .22 1.4.2 Glypican Functions in Drosophila .24 1.4.2.1 Regulation of Glypicans in Dpp Signaling 24 1.4.2.2 Regulation of Glypicans in Wingless Signaling 25 1.4.2.3 Regulation of Glypicans in Hedgehog Signaling 25 1.5 RNA Interference .26 1.6 Mediator Complex and its subunit 20 (Part II) 26 1.7 Function of Daughter of Sevenless in Drosophila (Par II) .28 ii 1.8 Objectives and Significance of This Thesis .28 Chapter II Materials and Methods .32 2.1 Molecular Work 32 2.1.1 PCR, Quantitative Real-time PCR, Inverse PCR and Primers 32 2.1.1.1 PCR .32 2.1.1.2 Quantitative Real-time PCR 33 2.1.1.3 Inverse PCR 33 2.1.1.4 PCR Primers 33 2.1.2 Construction of Recombinant Vectors 36 2.1.3 Strains and Growth Conditions 36 2.1.4 Transformation of E.coli DH5α Cells 37 2.1.4.1 Preparation of Competent Cells for Heat Shock Transformation 37 2.1.4.2 Heat Shock Transformation of DH5α 37 2.1.5 Plasmid DNA Preparation 38 2.1.5.1 Plasmid Miniprep 38 2.1.5.2 Plasmid Midiprep 39 2.1.6 RNA Extraction of Sorted Cells 39 2.1.7 Cell Death Determination 39 2.1.8 Synthesis of cDNA for PCR or Q-PCR .39 2.1.9 Amplification of RNA for Microarray Experiment (Part II: Med20) 40 2.1.10 Microarray Experiment 40 2.1.11 Single Fly DNA Preparation for Single Fly PCR 40 2.1.12 Fly DNA Preparation for PCR and Inverse PCR .41 2.2 Fluorescent Activated Cell Sorting (FACS) .41 2.2.1 Sample Preparation for FACS 41 2.2.2 Sorting of IGS cells by FACS 42 2.3 RNA In Situ Hybridization 42 2.3.1 RNA Probe Preparation 42 2.3.2 RNA In Situ Hybridization 43 iii 2.4 Immunofluorescence Staining and Confocal Microscope 44 2.4.1 Immunofluorescence Staining of Ovary 44 2.4.1.1 Fixation of Drosophila Ovaries 44 2.4.1.2 Antibody staining of Fixed Ovaries .44 2.4.1.3 Phosphorylated Extracellular Signal-regulated Kinase 1/2 (pErk 1/2) immunostaining 45 2.4.2 Confocal Image Processing 45 2.5 Fly Genetics 45 2.5.1 Fly Stocks 45 2.5.2 Mutagenesis through P-element Mediated Imprecise Excision 47 2.5.3 Homozygous Recombination .47 2.5.4 Drosophila Line Making through Chromosome Segregation 48 2.5.5 Germ Line Clone 48 2.5.5.1 Germline Clone Generation: 48 2.5.5.2 IGS Cell Clone Generation: 49 2.5.6 Germ Line Transformation 49 Chapter III Results (Part I) 51 EGFR signaling Restricts Germline Stem Cell Niche Activity in Drosophila Ovary 51 3.1 Involvement of Stet in EGFR Signaling in Drosophila Germaria .51 3.1.1 Introduction 51 3.1.2 Results 52 3.1.2.1 Functional Requirement of Stet in Germ Cells in Drosophila Germaria 52 3.1.2.2 Requirement of Multiple Membrane-tethered Ligands of EGFR in Drosophila Germaria .58 3.1.2.3 Requirement of Downstream Components of EGFR Signaling in IGS Cells 63 3.1.2.4 Requirement of Stet in Activation of EGFR/MAPK Signaling in IGS cells 68 iv 3.1.2.5 Requirement of Stet Function in GSCs 70 3.1.3 Discussions 76 3.2 Dpp Signaling Activity Affected by EGFR Signaling in Drosophila Germaria 78 3.2.1 Introduction 78 3.2.2 Results 78 3.2.2.1 Involvement of EGFR/MAPK Signaling in Repression of Dpp Signaling ouside the GSC Niche .78 3.2.2.2 Suppression of the Extra Spectrosome-containing Cells by dpphr56 .79 3.2.2.3 No ectopic Dpp transcripts detected in stet1A3 germaria 84 3.2.3 Discussions 86 3.3 Dally is Repressed by EGFR Signaling in IGS Cells in Drosophila Germaria 87 3.3.1 Introduction 87 3.3.2 Results 87 3.3.2.1 Requirement of dally Repression in IGS cells in Drosophila Germaria 87 3.3.2.2 Involvement of Dally in Dpp Signaling Activation .91 3.3.2.3 Occurrence of Dally Repression before Dpp Signaling Activation 94 3.3.3 Discussions 96 3.4 Conclusions 97 Chapter IV Results (Project II) .98 Somatic Control of Med20 in Drosophila Germaria 98 4.1 Requirement of med20 Function in Drosophila Germaria .98 4.1.1 Introduction 98 4.1.2 Results 98 4.1.2.1 Requirement of med20 for Germ Cell Differentiation in IGS cells 98 4.1.2.2 Discussions 100 4.2 Cell identity of Extra Spectrosome-Containing Cells in med20RNAi Germaria.102 4.2.1 Introduction 102 4.2.2 Results: Transient State of the Extra Spectrosome-containing Cells in v med20RNAi Germaria 102 4.3 Defective Cytoplasmic Extension in IGS cells in med20RNAi Germaria 103 4.4 Requirement of specific Components of Mediator Complex in IGS cells .106 4.5 Microarray Profiling of med20RNAi IGS Cells 106 4.5.1 Introduction 106 4.5.2 Results 109 4.6 Potential genes Regulated by Med20 .109 4.6.1 Introduction 109 4.6.2 Results 111 4.7 Conclusions 112 Chapter V Discussions 115 Cytoplasmic Extenstion Defect 115 Possible involvement of Dos in EGFR signaling 116 Part I: EGFR Signaling Restricts Germline Stem Cell Niche Activity in Drosophila 117 Involvement of Dally in other Morphogens 117 Function of Stet in other Types of Cells 118 stet Function in Larva Stage .118 stet Expression 119 Mechanism of EGFR/MAPK Signaling on Repression of dally 119 Interactions between the GSC Niche Activities 120 Part II: Somatic Control of Med20 on Germ Cells in Drosophila Germaria 120 dos is one Potential Target of Med20 120 A set of Potential Genes Affected by Med20 121 REFERENCES 123 PUBLICATIONS .130 vi LIST OF FIGURES AND TABLES FIGURES Figure 1.1 Anatomy of the Drosophila ovary and anterior germarium Figure 1.2 Schematic description of Dpp signaling in Drosophila 17 Figure 1.3 The canonical model of JAK-STAT signaling .18 Figure 1.4 Schematic EGFR signaling and its function 19 Figure 1.5 Schematic image of five EGFR ligands in Drosophila and intracellular cleavage and trafficking of Spi 20 Figure 1.6 Depiction of HSPGs associated with cell surface 23 Figure 3.1 Schematic image of deletion fragment in stet1A3 flies 54 Figure 3.2 Requirement of stet function in Drosophila germaria 56 Figure 3.3 Requirement of stet function in germ line .57 Figure 3.4 Rescue of the stet1A3 mutant phenotype by over-expression of stet in germ cells 59 Figure 3.5 Function analyses of EGFR membrane-tethered ligands in Drosophila germaria 62 Figure 3.6 Rescue phenotypes of stet1A3 germaria by individual EGFR secret ligands .64 Figure 3.7 Requirement of components of EGFR signaling in IGS cells in Drosophila germaria and cell death determination in stet1A3 germaria .67 Figure 3.8 plc-γ homozygous mutant phenotype, and knockdown phenotypes of pkb/akt and pi3k by RNA interference 69 Figure 3.9 Reduced pErk1/2 activity in stet1A3 germaria .71 Figure 3.10 Rescue of stet1A3 phenotype by over-expression of phl in IGS cells 72 Figure 3.11 Initial functional site of Stet in germ cells 75 Figure 3.12 Elevated Dpp signaling activity in stet1A3 and EGFRF24+RNAi germaria.81 Figure 3.13 Repressed bam expression in the extra Spectrosome-containing cells in stet1A3 germaria 82 vii Figure 3.14 Suppression of extra Spectrosome-containing cells by one copy removal of Dpp 83 Figure 3.15 RNA in situ hybridyzation of dpp transcript 85 Figure 3.16 Requirement of Dally repression by EGFR signaling in IGS cells shown by genetic data 89 Figure 3.17 Requirement of Dally repression by EGFR signaling in IGS cells shown by molecular data 90 Figure 3.18 Involvement of Dally in Dpp signaling activation 93 Figure 3.19 Occurrence of Dally repression before Dpp signaling activation 95 Figure 4.1 Functional analysis of med20 in Drosophila germaria 101 Figure 4.2 Cell identity of extra Spectrosome-containing cells in med20RNAi germaria 105 Figure 4.3 Phenotypes of med19RNAi and med27RNAi germaria 107 Figure 4.4 Sorting scope during FACS and sorted IGS cells 110 Figure 4.5 Q-PCR results of genes 113 Figure 4.6 Phenotypes of dosRNAi, cg8032RNAi and cg12340RNAi 114 viii TABLES Table 2.1 Primers for overexpression of secret form of keren and stet .34 Table 2.2 Primers for inverse PCR and excision determination PCR of stet and med20 34 Table 2.3 Primers for Q-PCR 35 Table 2.4 Primers for RNA in situ hybridyzation 36 Table 2.5 Antibodies used in this thesis study .44 Table 2.6 Fly stocks used in this thesis study 45 ix including GSCs, CBs, and cysts (called cytoplasmic extension), which is important for germline development Homeostasis between GSC self-renewal versus differentiation is very important for normal cytoplasmic extension Interestingly, the cytoplasmic extension was defective in all germaria bearing extra Spectrosome-containing cells, compared with wild type germaria Such cytoplasmic extension defect probably is due to cell over-proliferation of early germ cells, which disrupts homeostasis between GSC self-renewal vs differentiation Extra Spectrosome-containing cells in EGFR/MAPK signaling defective germaria resulted from ectopic Dpp signaling activation The ectopic Dpp signaling activation gives rise to extra Spectrosome-containing cells, which may be due to over-proliferation of GSC-like cells (Song et al., 2004) However, extra Spectrosome-containing cells could also result from CB-like cell proliferation in med20 knockdown (Figure 4.2I) Such over-proliferation of early germ cells caused germ cell overgrowth over cytoplamic extentsion of IGS cells, which ultimately could disrupt the coordination between early germ cells and IGS cells Possible involvement of Dos in EGFR signaling Both EGFR/MAPK signaling and med20 functional defect in IGS cells gave rise to extra Spectrosome-containing cells in germaria, but the cell identity of the extra Spectrosome-containing cells were different In EGFR/MAPK signaling defective germaria, the Spectrosome-containing cells were GSC-like, whereas those in med20RNAi germaria were in a transient state between GSCs and CBs Additionally, 116 pERK1/2 activity in med20RNAi germaria was similar to that in wild type germaria (data not shown) dos transcription was downregulated in med20RNAi IGS cells Dos can interact with Drk through its SH3 domain to exert its function in Sevenless and EGFR signalings (Feller et al., 2002) Whether Dos is involved in EGFR signaling in IGS cells is currently under investigation Part I: EGFR Signaling Restricts Germline Stem Cell Niche Activity in Drosophila This study has established the following sequence of events: 1) membrane-tethered EGFR ligand precursors are produced in germ cells, and Stet in germ cells likely cleave these precursors to form secreted active ligands; 2) the active ligands associate with EGFR in IGS cell surface to activate EGFR/MAPK signaling; 3) then the activated EGFR/MAPK signaling modulates dally expression in IGS cells; 4) IGS cells with low dally expression could not support a long range of Dpp transportation/stability, and consequently, CBs outside the GSC niche receive low/no DPP signal (niche activity) and undergo differentiation Thus Dpp function domain is confined within the GSC niche In conclusion, our results indicate that EGFR signaling plays a pivotal role in restricting the GSC niche activity through limiting Dally expression; GSCs also contribute to restrict the GSC niche activity Involvement of Dally in other Morphogens Our study results have demonstrated that repression of Dally in IGS cells is required for restriction of Dpp transportation/stability Because Dally is involved in several 117 morphogen distribution and subsequent signaling activation, whether repression of Dally is also required for restriction of other morphogen fuctions remains unclear in Drosophila germaria Function of Stet in other Types of Cells In EGFR signaling defective germaria, stet1A3 mutant gave rise to the most severe phenotype Extra Spectrosome-containg cell phenotype is observed in stet1A3 germaria within two days, which facilitates our study on it In addition, stet1A3 females died faster after eclosion, compared with both the male and wild type control Because mutations only in the ovary are not associated with lethality, thus this may be due to its key requirement in other tissues Additionally, knockdown of stet function in IGS cells gave rise to some defects in germ cell development However, the mechanism of stet function in IGS cells needs further investigation stet Function in Larva Stage Although there were no extra Spectrosome-containing cells just after eclosion, the possibility that Stet functions in larva stage cannot be excluded Stet is the only known Rhomboid protein expressed in Drosophila ovaries and one study has already shown that primordial germ cells express Spi to activate EGFR signaling for survival of the intermingled cells in Drosophila gonad In turn, the intermingle cells inhibit proliferation of the primordial germ cells (Gilboa and Lehmann, 2006) Thus, whether it is Stet that cleaves the Spi precursor in larva stage remains unclear 118 stet Expression It has been demonstrated that Stet in germ cells plays a role in restricting Dpp transportation/stability within the GSC niche This result dissects that early germ cells maintain homeostasis via restricting the GSC niche activity This study brings out an interesting question: how is stet expression regulated in early germ cells? Identification of the molecules regulating stet expression would give us a clearer picture of mechanism of stet Mechanism of EGFR/MAPK Signaling on Modulation of dally Another following up question is: how does EGFR signaling repress dally expression in IGS cells One recent paper has shown that dachsous and fat can negatively regulate dally expression via the Hippo signaling pathway in Drosophila imaginal wing disc (Baena-Lopez et al., 2008) Knockdown of dachsous, fat and components of the Hippo pathway was conducted However, germaria with compromised function in these genes in IGS cells not contain extra Spectrosome-containg cells These results suggest that dachsous, fat and the Hippo pathway in IGS cells are not involved in repressing dally expression in Drosophila germaria In the meantime, we tried to establish an in vitro system in Drosophila S2 cells to dissect how EGFR signaling represses dally expression However constitutively active form of EGFR gave rise to overexpression of dally, which was contrary to the situation in IGS cells in vivo, suggesting that regulation of EGFR/MAPK signaling on dally possibly is in a cell context-dependent manner Thus how EGFR signaling represses dally expression in 119 germaria needs further investigation Interactions between the GSC Niche Activities Our studies have shown that GSCs play an important role to trigger EGFR/MAPK signaling in IGS cells (escort stem cells and escort cells), which, therefore, restricts Dpp transportation/stability via repression of dally expression in IGS cells Thus EGFR/MAPK signaling is also one kind of the GSC niche activity Previous studies have demonstrated that Notch, Dpp and JAK-STAT signalings are associated with the niche activities All these niche-associated signals may function coordinately to control GSC behaviors For instance, it has been reported that JAK-STAT signaling positively regulates Dpp signaling (Lopez-Onieva et al., 2008; Wang et al., 2008) Interactions among these signals await further investigation Part II: Somatic Control of Med20 on Germ Cells in Drosophila Germaria Results from RNA interference-mediated screening revealed that somatic function of Med20 is required in CB-like cell differentiation And expression profiling of IGS cells demonstrated that Med20 regulates various genes expression, including dos dos is one Potential Target of Med20 According to the microarray data, the expression level of dos was reduced significantly in med20RNAi in IGS cell sample and the knockdown of dos in germaria resulted in formation of extra Spectrosome containing cells These evidence indicate 120 that dos might be one of the downstream targets of med20 However, the linkage between med20 and dos was unclear dos is involved in the signaling pathways regulated by receptor tyrosine kinases, such as Sevenless and EGFR signaling pathways (Bausenwein et al., 2000; Feller et al., 2002; Herbst et al., 1996; Raabe et al., 1996) And sev is required for formation of germline stem cell niche in Drosophila male gonad (Kitadate et al., 2007) However, the knockdown of sev did not show any phenotype in female, and the niche formation in med20RNAi and dosRNAi germaria was not affected So far, mechanism of Dos in IGS cells still remains unclear Thus, other associated proteins with Dos, including Corkscrew and Drk, need to be further examined In addition, the involvement of dos in other possible signalings, such as EGFR signaling remains unclear, which also needs to be investigated A set of Potential Genes Affected by Med20 Although previous studies show that mediator complex components specifically affect gene transcription, experimental results indicate that Med20 may affect sets of potential genes in Drosophila IGS cells Firstly, the experimental results provided evidences that dos might be the possible downstream target of med20 However, there were also differences between dos and med20RNAi phenotypes In the med20RNAi background, the germarium showed extra Spectrosome-containing cells at or days after eclosion However, in dosRNAi background, the formation of extra 121 Spectrosome-containing cell was only evident days after eclosion The differences between these two phenotypes are reasonable According to the microarray data, there were numerous genes affected in the med20RNAi IGS cell sample, and knocking down of several genes in IGS cells also resulted in the formation of ectopic Spectrosome-containing cells, reminiscent of the med20RNAi phenotype So it is possible that the med20RNAi phenotype resulted from the combination of more than one gene med20 could regulate a set of downstream genes Some of them maybe boost and some diminish the effect of med20RNAi dos gene could be part of the genes regulated by Med20 in IGS cells Also, to support that dos was the real downstream target of med20, the restoring dos in med20RNAi background are necessary If the over expression of dos could fully or partially rescue the med20RNAi phenotype, the 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Titmeng Lim, Yu Cai Somatic Control of Med20 on the Differentiation of Early Germ Cells in Drosophila Germaria (manuscript in preparation); 130 ... about interactions between early germ cells (including GSC, CB and early cysts) and inner germarial sheath (IGS) cells outside the niche In my thesis study, I would address the interactions between. .. on interactions between early germ cells and inner germarial sheath cells (IGS cells) during Drosophila oogenesis Two aspects of signals are studied: one, signals from early germ cells to IGS cells. .. between early germ cells, including GSCs, and IGS cells in Drosophila ovary 1.1 GSCs and the GSC Niche in Drosophila Ovary Female GSCs are closely associated with the surrounding supporting cells,