Investigating the Co-Regulatory Role of Midline and Extramacrochaetae in Regulating Eye Development and Vision in Drosophila Melanogaster
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The University of Southern Mississippi The Aquila Digital Community Honors Theses Honors College Spring 5-2014 Investigating the Co-Regulatory Role of Midline and Extramacrochaetae In Regulating Eye Development and Vision in Drosophila melanogaster Lillian M. Forstall University of Southern Mississippi Follow this and additional works at: https://aquila.usm.edu/honors_theses Part of the Developmental Biology Commons Recommended Citation Forstall, Lillian M., "Investigating the Co-Regulatory Role of Midline and Extramacrochaetae In Regulating Eye Development and Vision in Drosophila melanogaster" (2014). Honors Theses. 236. https://aquila.usm.edu/honors_theses/236 This Honors College Thesis is brought to you for free and open access by the Honors College at The Aquila Digital Community. It has been accepted for inclusion in Honors Theses by an authorized administrator of The Aquila Digital Community. For more information, please contact [email protected]. The University of Southern Mississippi Investigating the Co-Regulatory Role of midline and extramacrochaetae In Regulating Eye Development and Vision in Drosophila melanogaster by Lillian Forstall A Thesis Submitted to the Honors College of The University of Southern Mississippi in Partial Fulfillment of the Requirements for the Degree of Bachelor of Science in the Department of Biological Sciences May 2014 ii Approved by _________________________________ Dr. Sandra Leal, Ph.D., Thesis Adviser Assistant Professor of Biology _________________________________ Dr. Shiao Wang, Ph.D., Interim Chair Department of Biological Sciences _________________________________ Dr. David R. Davies, Ph.D., Dean Honors College iii Abstract The Honors thesis research focused on the roles of extramacrochaetae and midline in regulating eye development and the vision of Drosophila melanogaster. It is known from previous studies that extramacrochaetae (emc) and midline (mid) independently regulate the formation of ommatidial units in the Drosophila compound eye. However, the thesis focuses on the interaction of these two genes and their co-dependent roles in regulating eye development. This study also attempts to explain the recovered formation of ommatidial units and interommatidial bristles when the expression of both of these genes is reduced and whether flies doubly mutant for these genes have recovered phototactic ability. Specific genotypes of flies were subjected to larval and adult phototaxis assays to assay their phototactic ability. A Western analysis was performed on extramacrochaetae mutants, midline mutants, and wild-type flies to determine whether the Emc and Mid proteins interacted in a co-regulatory fashion within developing larval tissues. The larval phototaxis assays revealed a slight decrease in photoreception in the mid- RNAi larvae when compared to the wild-type larvae. However the data was not conclusive to definitively determine if the mid-RNAi mutants displayed a significant decrease in photoreceptive ability. The adult phototaxis assays were more definitive than the larval assays. The emc1 flies displayed a slight decrease in photoreceptive ability. Both the mid- RNAi and the flies doubly mutant for midGA174 and emc1 displayed a significant decrease in photoreceptive ability. The Western blot and immunofluorescence studies revealed an interaction between mid and emc, and the future nature of this interaction will be resolved in greater detail Key Terms: Fruit fly, compound eye, gene regulation, gene expression, Honors College. iv Dedication Barbara, Mark and John Forstall Without whom I could not conquer my greatest hopes and fears. You are the perfect support system. v Acknowledgements I would like to thank my thesis advisor, Dr. Sandra Leal, for guiding me through the process of becoming a true scientific researcher by making genetics and developmental biology come alive. This thesis would not be possible had it not been for her creative spirit and willingness to pass on her knowledge to others. Thank you for everything you do. I would also like to thank Ken Buford, without whom I would not have completed all of my research, and without whom I might have lost a finger. Additionally, I would like to thank the Honors College for giving me the opportunity to better myself through research and challenging me with accelerated classes. Without the Honors College, I would not have achieved all the goals I set when I set foot on this campus. vi Table of Contents List of Figures ........................................................................................................................viii List of Tables .........................................................................................................................ix List of Abbreviations .............................................................................................................x Chapter 1: Introduction .........................................................................................................1 Chapter 2: Review of Literature ...........................................................................................4 Drosophila melanogaster...........................................................................................4 extramacrochaetae and midline .................................................................................5 Ommatidia and Eye Formation ..................................................................................5 Chapter 3: Methodology .......................................................................................................7 Larval Phototaxis Assay ............................................................................................7 Adult Phototaxis Assay ..............................................................................................8 Immunofluorescence ..................................................................................................9 Western Blot for Protein Interaction ..........................................................................11 Chapter 4: Results .................................................................................................................14 Larval Phototaxis Assay ............................................................................................14 Female Adult Phototaxis Assay .................................................................................15 Male Adult Phototaxis Assay.....................................................................................16 Western Blot ..............................................................................................................17 Immunofluorescence ..................................................................................................19 Chapter 5: Discussion ...........................................................................................................19 Chapter 6: Conclusion...........................................................................................................22 Literature Cited ......................................................................................................................24 vii List of Figures Figure 1: Wild-type, midline Eye Comparison .....................................................................1 Figure 2: Wild-type, midline, extramacrochaetae Eye Comparison ....................................2 Figure 3: Wild-type and midline Immunofluorescence ........................................................3 Figure 4: Photoreceptor Formation .......................................................................................7 Figure 5: Larval Phototaxis Assay Plate ...............................................................................8 Figure 6: Adult Phototaxis Assay Apparatus ........................................................................9 Figure 7: Third-Instar Larval Phototaxis ..............................................................................14 Figure 8: Adult Female Phototaxis Results ..........................................................................15 Figure 9: Adult Male Phototaxis Results ..............................................................................16 Figure 10: Western Blot for H15 ..........................................................................................17 Figure 11: Western Blot for Emc ..........................................................................................18 Figure 12: Immunofluorescence Study .................................................................................19 viii List of Tables Table 1: Separating Gel Solution ...........................................................................................11 Table 2: Stacking Gel Solution ..............................................................................................12 ix List of Abbreviations Central nervous system .......................................................................................................CNS Double mutant .....................................................................................................................DM Inhibitor of differentiation ..................................................................................................ID extramacrochaetae ..............................................................................................................emc midline.................................................................................................................................mid