U N V I ìo Identifying the roles of dead rínger in the Drosophilø eye A thesis submitted for the degree of Doctor of Philosophy Jane Sibbons, B.Sc. (Hons.) School of Molecular and Biomedical Sciences, Genetics discipline Centre for the Molecular Genetics of Development The University of Adelaide September,2004 Table of Contents Table of Contents I Table of Figures IV Table of Tables iv Abstract V Declaration v11 Acknowledgments ix Chapter 1: Introduction I 1.1 Transcription factors and animal development I LI.1 The ARIDfamily of transcriptionfactors I 1.2 The ARID family of proteins 2 1.2.1 The Jumonji group of proteins 2 L2.2 Other core ARID family members 3 1.2.3 The extended Ak[D group of proteins 3 1.2.3.1 Bright 4 L2.3.2 Bright Dead ringer Protein 4 ].2.3.3 DRILl/E2FBP1 4 L2.3.4 CFI-I 5 1.3 ARID domain structure 5 1.4 Embryonic expression of dri and, the known target genes of Dri regulation _ 7 1.5 Differentiation of the components of the adult 8 1.6 Axon connections between the developing eye and brain 11 L6.1 R(-R6 axons terminate in the lamina 13 L6.1 .1 RI -R6 axon termination 13 L6.1.2 Neuron dffirentiation within the lamina 16 L6. 1. 3 Neural superposition t7 1.6.2 R7 and R8 axons terminate in the medulla I7 L6.3 Genetic Screens undertaken to identify proteins requiredfor the conect targeting of R axons I9 L 6. 3. I The immunohistochemical screen 19 1.6.3.2 The behavioural screen 20 1.7 Visual transduction 2l 1.7.1 Regulation of the rhodopsin genes 22 L7.1.1 Regulation of rhodopsin in RI to R6 23 1.7.L2 Regulation of rhodopsin expression in R7 and R8 23 1.7.2 Light activation of the visual transduction pathway 27 L7.3 Termination of the light pathway 27 1.7.4 Degeneration of the rltabdomeres 30 1.8 Aims and approaches of this thesis 32 Chapter 2: Methods and Materials 33 2.1 Abbreviations 33 2.2 Materials 33 2.2.1 Molecular weight markers i3 I 2.2.2 Media, Buffers and Solutions 33 2.2.3 Fly stock 34 2.2.4 Antibodies 35 2.2.5 Primers 37 2.3 Methods 37 2.3.1 Light and dark reared conditions for Drosophila 37 2.3.2 Sectioning 37 2. 3. 2. I Tangential sectioning 37 2.3.2.2 Adult head sectioning 37 2.3.2.2.1DAB staining 37 2.3 .2.2.2 Fluorescent staining 38 2. 3. 3 Scanning electron micrograph 38 2. 3.4 Antibody Staining 38 2. 3. 5 Gel electrophoresis 39 2. 3. 5. 1 KNase-free Agarose gel electrophoresis 39 2.3.5.2 SDS PAGE gel 39 2. 3.6 I(estern analysis 40 2.3.6.1 Semi-quantitative analysis from Western blots 40 2.3.7 Collection of materialfor Real time PCR analysis 40 2.3.8 Extraction of total RNA 40 2.3.9 Real time PCR 41 2.3.9.1 cDNA synthesis 41 2.3.9.2 Real time PCR 41 Chapter 3: The expression of deød ringer in the eye 43 3.1 Introduction 43 3.2 Results 43 3.2.1 Dri is expressed in RL-R6 and R8 cells during the third larval instar 43 3.2.2 Expression of Dri in pupal stages 45 3.2.3 Adult expression of Dri 49 3.3 Discussion 51 3.3.I The expression of Dri during the third larval instar 5I 3.3.2 Expression of Dri during pupal and adult stages 51 3.3.3 The expression patterns and possible Dri function 52 Chapter 4: Role of deød ringer in the developing eye 55 4.1 Introduction 55 4.2 Results 55 4.2.1 The loss of Dri in the eye results in blindness 55 4.2.2 The loss of Dri does not affect the dffirentiation or fate ofthe photoreceptor cells 57 4.2.3 The monopolar neurons are differentiated in the absence of Dri 63 4.2.4 Axon projections are disrupted in the absence of Dri 63 4.2.5 R8 axons are targeted to the correct layer in the absence of Dri 65 4.2.6 R1-R6 axons pass through to the medulla in dri mutant eye mosaics 69 4.2.7 Glial cell migration is not the reasonfor the Rl-R6 targeting defects in dri 69 mutant eye mosaics - 4.2.8 Dri does not regulate runt expression 73 4.2.9 The adult axonal defects are mild in dri mutant mosaic eyes 73 4.3 Discussion 77 4.3.1 Dri is requiredfor Rl-R6 axons to terminate in the lamina 77 4.3.2 Possible targets for Dri transcriptional regulation 78 ll 4.3.3 Conclusions 79 Chapter 5: The role of dead ringer in the visual system 81 5.1 Introduction 81 5.2 Results 82 5.2.1 The Rhl protein level is significantly reduced in dri mutant eye mosaics _ 82 5.2.2 Dri does not directly regulate ninaE transcription 85 5.2.3 Rhabdomeres in dri mutant eye mosaics degenerate in an age- and light- dependent manner 86 5.2. 3. 1 FRT42D control.flies 93 5.2.3.2 Dark-reared dri mutant eye mosaicflies 93 5.2.3.3 Light-reared dri mutant eye mosaicflies 94 5.3 Discussion 99 5.3.1 dri is requiredþr normal RhI levels andfor normal rhabdomeres structure and stability 99 5.3.2 At which stage in the termination of the visual transduction pathway could dri act? I0I 5.3.3 Other possible roles for dri r06 5.3.4 Conclusions 106 Chapter 6: Final Discussion 107 6.1 Introduction 107 6.2Dri expression at different stages of eye development 107 6.3 The role of dri dwing the third larval Ínstar stage 107 6.3.1 Dri appears to define a new transcriptional regulatory component of p hot or e c ep t or ax ono gen es is 108 6.3.2 Dri is requiredfor conect growth cone shape in R8 cells 109 6.4 The role of dri in the adult eye 110 6.4.1 Rhodopsin l fails to accumulate in dri mutant eye mosaics 110 6.4.2 Light-dependent degeneration in dri mutant eyes 111 6.4.3 Does dri play an evolutionarily conserved role in eye development? _ 112 6.5 Conclusion ll2 Appendices 115 References 119 ul Table of Figures Figure 1.1 The structure of the ARID domain in Drr 6 Figure l.2The differentiation and components of the Drosophila eye.............50 Figure 1.3 The termination of the R ce1l axons in the optic lobe.......................12 Figure 1.4 Regulation of the different Rhodopsins 26 Figure 1.5 The visual pathway inDrosophila....... 28 Figure 3.1 Expression of Dri in eye development.......... ......44 Figure 3.2Bxpression of Dri during pupal stages ...............46 Figure 3.3 Dri is down regulated in R8 cells during pupal development ..........48 Figure 3.4 Adult expression of Dri.. 50 Figure 4.I Creating somatic mutant clones that spanned the entire eye s6 Figure 4.2 dri mutant eye mosaic animals are blind 58 Figure 4.3 The R cells are specified normally in dri mutant eye mosaics.........60 Figure 4.4 The structure of the dri mosaic adult eye is relatively normal ... .. 62 Figure 4.5 The specification of the monopolar neurons is unaffected in the absence of Dri .....64 Figure 4.6 R cell axon targeting indri mutant eye mosaics is disrupted...........66 Figure 4.7 R8 axon growth cone shape is disrupted in dri mutant eye mosaics 68 Figure 4.8 A subset of R1-R6 axons terminate in the medulla 70 Figure 4.9 Lamina glial cell migration occurs normally in the absence of Drt .72 Figure 4.10 runt expression is unaffected by the loss of Dri 74 Figure 4.1 1 Loss of Dri results in mild adult axonal defects 76 Figure 5.1 Rhl protein levels are reduced in dri mutant eye mosaic flies......... 83 Figure 5.2 R cells undergo age- and light-dependent degeneration in dri mutant eye mosalcs 88 Figure 5.3 R cells degenerate in an age- and light-dependent manner tn dri mutant mosaic adult eyes 90 Figure 5.4 Rhl proteins do not change in light or dark reared conditions.........96 Figure 5.5 R cells degenerate in an age- and light-dependent manner in dri mutant eye mosaic adult eyes 98 Figure 5.6 Degeneration holes appear tn dri mutant eye mosaic sections....... 100 Figure 5.7 Potential Dri targets in the light pathway r04 Table of Tables Table 1: The relative expression of ninaE mRNA compared to rp49 in dri mutant eye mosaic heads compared to FRT42D control' 86 Table 2: The phenotypes of ommatidia scored tn FRT42D control and dri mutant eye mosaics at newly eclosed, 4 and 6 weeks of age from flies reared in both light and dark conditions. ..... 87 IV Abstract The Drosophila Dead ringer (Dri) protein is a founding member of the ARID family of transcription factors, which play important roles in many important biological processes. dri is essential for Drosophila development, with mutations in dri resulting in embryonic lethality. Although some of the roles of Dri have been identified in the embryo, little is known about its roles in later stages of development.