Advertisement

Analysis of the Drosophila Compound Eye with Light and Electron Microscopy

  • Monalisa Mishra
  • Elisabeth Knust
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1834)

Abstract

The Drosophila compound eye is composed of about 750 units, called ommatidia, which are arranged in a highly regular pattern. Eye development proceeds in a stereotypical fashion, where epithelial cells of the eye imaginal discs are specified, recruited, and differentiated in a sequential order that leads to the highly precise structure of an adult eye. Even small perturbations, for example in signaling pathways that control proliferation, cell death, or differentiation, can impair the regular structure of the eye, which can be easily detected and analyzed. In addition, the Drosophila eye has proven to be an ideal model for studying the genetic control of neurodegeneration, since the eye is not essential for viability. Several human neurodegeneration diseases have been modeled in the fly, leading to a better understanding of the function/misfunction of the respective gene. In many cases, the genes involved and their functions are conserved between flies and human. More strikingly, when ectopically expressed in the fly eye some human genes, even those without a Drosophila counterpart, can induce neurodegeneration, detectable by aberrant phototaxis, impaired electrophysiology, or defects in eye morphology and retinal histology. These defects are often rather subtle alteration in shape, size, or arrangement of the cells, and can be easily scored at the ultrastructural level. This chapter aims to provide an overview regarding the analysis of the retina by light and electron microscopy.

Key words

Drosophila melanogaster Light microscopy Deep pseudopupil Electron microscopy Cryolabeling Whole mount Compound eye 

Notes

Acknowledgments

We thank Michaela Rentsch for help with the electron micrographs; Franziska Friedrich for help in preparing Figs. 3, 4, and 5; Nagananda Gurudev for the figure of the optical neutralization; and Sarita Hebbar for helpful comments. Work of E. K. is supported by the Max-Planck Society (MPG).

References

  1. 1.
    Wolff T, Ready DF (1991) Cell death in normal and rough eye mutants of Drosophila. Development 113:825–839PubMedGoogle Scholar
  2. 2.
    Baumann O, Lutz K (2006) Photoreceptor morphogenesis in the Drosophila compound eye: R1–R6 rhabdomeres become twisted just before eclosion. J Comp Neurol 498:68–79CrossRefGoogle Scholar
  3. 3.
    Zuker, C. S., ., Cowman, A. F., and Rubin, G. M. (1985) Isolation and structure of a rhodopsin gene from D. melanogaster, Cell 40, 851-858CrossRefGoogle Scholar
  4. 4.
    O'Tousa JE, Baehr W, Martin RL, Hirsh J, Pak WL, Applebury ML (1985) The Drosophila ninaE gene encodes an opsin. Cell 40:839–850CrossRefGoogle Scholar
  5. 5.
    Montell C, Jones K, Zuker C, Rubin G (1987) A second opsin gene expressed in the ultraviolet-sensitive R7 photoreceptor cells of Drosophila melanogaster. J Neurosci 7:1558–1566CrossRefGoogle Scholar
  6. 6.
    Zuker CS, Montell C, Jones K, Laverty T, Rubin GM (1987) A rhodopsin gene expressed in photoreceptor cell R7 of the Drosophila eye: homologies with other signal-transducing molecules. J Neurosci 7:1550–1557CrossRefGoogle Scholar
  7. 7.
    Feiler R, Bjornson R, Kirschfeld K, Mismer D, Rubin GM, Smith DP, Socolich M, Zuker CS (1992) Ectopic expression of ultraviolet-rhodopsins in the blue photoreceptor cells of Drosophila: visual physiology and photochemistry of transgenic animals. J Neurosci 12:3862–3868CrossRefGoogle Scholar
  8. 8.
    Salcedo E, Huber A, Henrich S, Chadwell LV, Chou WH, Paulsen R, Britt SG (1999) Blue- and green-absorbing visual pigments of Drosophila: ectopic expression and physiological characterization of the R8 photoreceptor cell-specific Rh5 and Rh6 rhodopsins. J Neurosci 19:10716–10726CrossRefGoogle Scholar
  9. 9.
    Montell C (2012) Drosophila visual transduction. Trends Neurosci 35:356–363CrossRefGoogle Scholar
  10. 10.
    Harris WA, Stark WS, Walker JA (1976) Genetic dissection of the photoreceptor system in the compound eye of Drosophila melanogaster. J Physiol 256:415–439CrossRefGoogle Scholar
  11. 11.
    Reinke R, Krantz DE, Yen D, Zipursky SL (1988) Chaoptin, a cell surface glycoprotein required for Drosophila photoreceptor cell morphogenesis, contains a repeat motif found in yeast and human. Cell 52:291–301CrossRefGoogle Scholar
  12. 12.
    Tomlinson A, Bowtell DD, Hafen E, Rubin GM (1987) Localization of the sevenless protein, a putative receptor for positional information, in the eye imaginal disc of Drosophila. Cell 51:143–150CrossRefGoogle Scholar
  13. 13.
    Campos-Ortega JA, Jürgens G, Hofbauer A (1979) Cell clones and pattern formation: studies on sevenless, a mutant of Drosophila melanogaster. Wilhelm Roux’s Arch Dev Biol 186:27–50CrossRefGoogle Scholar
  14. 14.
    Tomlinson A, Kimmel BE, Rubin GM (1988) Rough, a Drosophila homeobox gene required in photoreceptors R2 and R5 for inductive interactions in the developing eye. Cell 55:771–784CrossRefGoogle Scholar
  15. 15.
    Baker NE, Rubin GM (1989) Effect on eye development of dominant mutations in Drosophila homologue of the EGF receptor. Nature 340:150–153CrossRefGoogle Scholar
  16. 16.
    Miyamoto H, Nihonmatsu I, Kondo S, Ueda R, Togashi S, Hirata K, Ikegami Y, Yamamoto D (1995) Canoe encodes a novel protein containing a GLGF/DHR motif and functions with Notch and scabrous in common developmental pathways in Drosophila. Genes Dev 9:612–625CrossRefGoogle Scholar
  17. 17.
    Basler K, Christen B, Hafen E (1991) Ligand-independent activation of the sevenless receptor tyrosine kinase changes the fate of cells in the developing Drosophila eye. Cell 64:1069–1081CrossRefGoogle Scholar
  18. 18.
    Grzeschik N, Knust E (2005) IrreC/rst-mediated cell sorting during Drosophila pupal eye development depends on proper localisation of DE-cadherin. Development 132:2035–2045CrossRefGoogle Scholar
  19. 19.
    Yang Y, Ballinger D (1994) Mutations in calphotin, the gene encoding a Drosophila photoreceptor cell-specific calcium-binding protein, reveal roles in cellular morphogenesis and survival. Genetics 138:413–421PubMedPubMedCentralGoogle Scholar
  20. 20.
    Mishra M, Oke A, Lebel C, McDonald EC, Plummer Z, Cook TA, Zelhof AC (2010) Pph13 and orthodenticle define a dual regulatory pathway for photoreceptor cell morphogenesis and function. Development 137:2895–2904CrossRefGoogle Scholar
  21. 21.
    Zelhof AC, Koundakjian E, Scully AL, Hardy RW, Pounds L (2003) Mutation of the photoreceptor specific homeodomain gene Pph13 results in defects in phototransduction and rhabdomere morphogenesis. Development 130:4383–4392CrossRefGoogle Scholar
  22. 22.
    Li BX, Satoh AK, Ready DF (2007) Myosin V, Rab11 and dRip11 direct apical secretion and cellular morphogenesis in Drosophila photoreceptor cells. J Cell Biol 177:659–669CrossRefGoogle Scholar
  23. 23.
    Muschalik N, Knust E (2011) Increased levels of the cytoplasmic domain of Crumbs repolarise developing Drosophila photoreceptors. J Cell Sci 124:3715–3725CrossRefGoogle Scholar
  24. 24.
    Richard M, Grawe F, Knust E (2006) DPATJ plays a role in retinal morphogenesis and protects against light-dependent degeneration of photoreceptor cells in the Drosophila eye. Dev Dyn 235:895–907CrossRefGoogle Scholar
  25. 25.
    Johnson K, Grawe F, Grzeschik N, Knust E (2002) Drosophila Crumbs is required to inhibit light-induced photoreceptor degeneration. Curr Biol 12:1675–1680CrossRefGoogle Scholar
  26. 26.
    Hong Y, Ackerman L, Jan LY, Jan Y-N (2003) Distinct roles of Bazooka and Stardust in the specification of Drosophila photoreceptor membrane architecture. Proc Natl Acad Sci U S A 100:12712–12717CrossRefGoogle Scholar
  27. 27.
    Berger S, Bulgakova NA, Grawe F, Johnson K, Knust E (2007) Unravelling the genetic complexity of Drosophila stardust during photoreceptor morphogenesis and prevention of light-induced degeneration. Genetics 176:2189–2200CrossRefGoogle Scholar
  28. 28.
    Pellikka M, Tanentzapf G, Pinto M, Smith C, McGlade CJ, Ready DF, Tepass U (2002) Crumbs, the Drosophila homologue of human CRB1/RP12, is essential for photoreceptor morphogenesis. Nature 416:143–149CrossRefGoogle Scholar
  29. 29.
    Pham H, Yu H, Laski FA (2008) Cofilin/ADF is required for retinal elongation and morphogenesis of the Drosophila rhabdomere. Dev Biol 318:82–91CrossRefGoogle Scholar
  30. 30.
    Matsuo T, Takahashi K, Suzuki E, Yamamoto D (1999) The Canoe protein is necessary in adherens junctions for development of ommatidial architecture in the Drosophila compound eye. Cell Tissue Res 298:397–404CrossRefGoogle Scholar
  31. 31.
    Husain N, Pellikka M, Hong H, Klimentova T, Choe K-M, Clandinin TR, Tepass U (2006) The agrin/perlecan-related protein eyes shut is essential for epithelial lumen formation in the Drosophila retina. Dev Cell 11:483–493CrossRefGoogle Scholar
  32. 32.
    Zelhof AC, Hardy RW, Becker A, Zuker CS (2006) Transforming the architecture of compound eyes. Nature 443:696–699CrossRefGoogle Scholar
  33. 33.
    Cheli VT, Daniels RW, Godoy R, Hoyle DJ, Kandachar V, Starcevic M, Martinez-Agosto JA, Poole S, DiAntonio A, Lloyd VK, Chang HC, Krantz DE, Dell’Angelica EC (2010) Genetic modifiers of abnormal organelle biogenesis in a Drosophila model of BLOC-1 deficiency. Hum Mol Genet 19:861–878CrossRefGoogle Scholar
  34. 34.
    Pulipparacharuvil S, Akbar MA, Ray S, Sevrioukov EA, Haberman AS, Rohrer J, Kramer H (2005) Drosophila Vps16A is required for trafficking to lysosomes and biogenesis of pigment granules. J Cell Sci 118:3663–3673CrossRefGoogle Scholar
  35. 35.
    Wu CF, Wong F (1977) Frequency characteristics in the visual system of Drosophila: genetic dissection of electroretinogram components. J Gen Physiol 69:705–724CrossRefGoogle Scholar
  36. 36.
    Hardie RC, Postma M (2008) Phototransduction in microvillar photoreceptors of Drosophila and other invertebrates. In: Basbaum AI, Kaneko A, Shephard GM, Westheimer G (eds) The senses: a comprehensive reference. Academic Press, San Diego, pp 77–130CrossRefGoogle Scholar
  37. 37.
    Pak WL (2010) Why Drosophila to study phototransduction? J Neurogenet 24:55–66CrossRefGoogle Scholar
  38. 38.
    Pak WL, Grossfield J, Whiten V (1969) Non- phototactic mutants in a study of vision of Drosophila. Nature 222:351–354CrossRefGoogle Scholar
  39. 39.
    Hotta Y, Benzer S (1969) Abnormal electroretinograms in visual mutants of Drosophila. Nature 222:354–356CrossRefGoogle Scholar
  40. 40.
    Heisenberg M (1971) Isolation of mutants lacking the optomotor response. Dros Inf Serv 112:65–93Google Scholar
  41. 41.
    Heisenberg M (1997) Genetic approach to neuroethology. BioEssays 19:1065–1073CrossRefGoogle Scholar
  42. 42.
    Franceschini N (1972) Pupil and Pseudopupil in the compound eye of Drosophila. In: Wehner R (ed) Information processing in the visual systems of arthropods. Springer, Berlin, pp 75–82CrossRefGoogle Scholar
  43. 43.
    Steele F, O'Tousa JE (1990) Rhodopsin activation causes retinal degeneration in Drosophila rdgC mutant. Neuron 4:883–890CrossRefGoogle Scholar
  44. 44.
    Pichaud F, Desplan C (2001) A new visualization approach for identifying mutations that affect differentiation and organization of the Drosophila ommatidia. Development 128:815–826PubMedGoogle Scholar
  45. 45.
    Meyer NE, Joel-Almagor T, Frechter S, Minke B, Huber A (2006) Subcellular translocation of the eGFP-tagged TRPL channel in Drosophila photoreceptors requires activation of the phototransduction cascade. J Cell Sci 119:2592–2603CrossRefGoogle Scholar
  46. 46.
    Pandey UB, Nichols CD (2011) Human disease models in Drosophila melanogaster and the role of the fly in therapeutic drug discovery. Pharmacol Rev 63:411–436CrossRefGoogle Scholar
  47. 47.
    Whitworth AJ (2011) Drosophila models of Parkinson’s disease. Adv Genet 73:1–50PubMedGoogle Scholar
  48. 48.
    Ambegaokar SS, Roy B, Jackson GR (2010) Neurodegenerative models in Drosophila: polyglutamine disorders, Parkinson disease, and amyotrophic lateral sclerosis. Neurobiol Dis 40:29–39CrossRefGoogle Scholar
  49. 49.
    St. Johnston D (2002) The art and design of genetic screens: Drosophila melanogaster. Nat Rev Genet 31:176–188CrossRefGoogle Scholar
  50. 50.
    Wang T, Montell C (2007) Phototransduction and retinal degeneration in Drosophila. Pflügers Arch 454:821–847CrossRefGoogle Scholar
  51. 51.
    Fernandez-Funez P, Nino-Rosales ML, de Gouyon B, She WC, Luchak JM, Martinez P, Turiegano E, Benito J, Capovilla M, Skinner PJ, McCall A, Canal I, Orr H, Zoghbi HY, Botas J (2000) Identification of genes that modify ataxin-1-induced neurodegeneration. Nature 408:101–106CrossRefGoogle Scholar
  52. 52.
    Cook T, Zelhof A, Mishra M, Nie J (2011) 800 facets of retinal degeneration. Prog Mol Biol Transl Sci 100:331–368CrossRefGoogle Scholar
  53. 53.
    Lu B (2009) Recent advances in using Drosophila to model neurodegenerative diseases. Apoptosis 14:1008–1020CrossRefGoogle Scholar
  54. 54.
    Bonini NM, Fortini ME (2002) Applications of the Drosophila retina to human disease modeling. In: Moses K (ed) Drosophila eye development. Springer, Heidelberg, pp 257–271CrossRefGoogle Scholar
  55. 55.
    Ugur B, Chen K, Bellen HJ (2016) Drosophila tools and assays for the study of human diseases. Dis Model Mech 9:235–244CrossRefGoogle Scholar
  56. 56.
    Xiong B, Bellen HJ (2013) Rhodopsin homeostasis and retinal degeneration: lessons from the fly. Trends Neurosci 36:652–660CrossRefGoogle Scholar
  57. 57.
    Millburn GH, Crosby MA, Gramates LS, Tweedie S, FlyBase C (2016) FlyBase portals to human disease research using Drosophila models. Dis Model Mech 9:245–252CrossRefGoogle Scholar
  58. 58.
    Tepass U, Knust E (1993) Crumbs and stardust act in a genetic pathway that controls the organization of epithelia in Drosophila melanogaster. Dev Biol 159:311–326CrossRefGoogle Scholar
  59. 59.
    Izaddoost S, Nam S-C, Bhat MA, Bellen HJ, Choi K-W (2002) Drosophila Crumbs is a positional cue in photoreceptor adherens junctions and rhabdomeres. Nature 416:178–183CrossRefGoogle Scholar
  60. 60.
    Bhat MA, Izaddoost S, Lu Y, Cho KO, Choi KW, Bellen HJ (1999) Discs Lost, a novel multi-PDZ domain protein, establishes and maintains epithelial polarity. Cell 96:833–345CrossRefGoogle Scholar
  61. 61.
    Bachmann A, Schneider M, Grawe F, Theilenberg E, Knust E (2001) Drosophila Stardust is a partner of Crumbs in the control of epithelial cell polarity. Nature 414:638–643CrossRefGoogle Scholar
  62. 62.
    Hong Y, Stronach B, Perrimon N, Jan LY, Jan YN (2001) Drosophila Stardust interacts with Crumbs to control polarity of epithelia but not neuroblasts. Nature 414:634–638CrossRefGoogle Scholar
  63. 63.
    Bulgakova NA, Rentsch M, Knust E (2010) Antagonistic functions of two Stardust isoforms in Drosophila photoreceptor cells. Mol Biol Cell 21:3915–3925CrossRefGoogle Scholar
  64. 64.
    Bulgakova NA, Kempkens Ö, Knust E (2008) Multiple domains of Drosophila Stardust differentially mediate localisation of the Crumbs/Stardust complex during photoreceptor development. J Cell Sci 121:2018–2026CrossRefGoogle Scholar
  65. 65.
    Oda H, Uemura T, Harada Y, Iwai Y, Takeichi M (1994) A Drosophila homolog of cadherin associated with armadillo and essential for embryonic cell-cell adhesion. Dev Biol 165:716–726CrossRefGoogle Scholar
  66. 66.
    Riggleman B, Schedl P, Wieschaus E (1990) Spatial expression of the Drosophila segment polarity gene armadillo is posttranscriptionally regulated by wingless. Cell 63:549–560CrossRefGoogle Scholar
  67. 67.
    Karagiosis SA, Ready DF (2004) Moesin contributes an essential structural role in Drosophila photoreceptor morphogenesis. Development 131:725–732CrossRefGoogle Scholar
  68. 68.
    Satoh AK, Li BX, Xia H (2008) Calcium-activated myosin V closes the Drosophila pupil. Curr Biol 18:951–955CrossRefGoogle Scholar
  69. 69.
    Lebovitz RM, Takeyasu K, Fambrough DM (1989) Molecular characterization and expression of the (Na+/K+)-ATPase alpha-subunit in Drosophila melanogaster. EMBO J 8:193–201CrossRefGoogle Scholar
  70. 70.
    Yasuhara JC, Baumann O, Takeyasu K (2000) Localization of Na/K-ATPase in developing and adult Drosophila melanogaster photoreceptors. Cell Tissue Res 300:239–249CrossRefGoogle Scholar
  71. 71.
    Blochlinger K, Bodmer R, Jan LY, Jan YN (1990) Patterns of expression of cut, a protein required for external sensory organ development in wild-type and cut mutant Drosophila embryos. Genes Dev 4:1322–1331CrossRefGoogle Scholar
  72. 72.
    Zipursky SL, Venkatesh TR, Teplow DB, Benzer S (1984) Neuronal development in the Drosophila retina: monoclonal antibodies as molecular probes. Cell 36:15–26CrossRefGoogle Scholar
  73. 73.
    Yano H, Yamamoto-Hino M, Goto S (2009) Spatial and temporal regulation of glycosylation during Drosophila eye development. Cell Tissue Res 336:137–147CrossRefGoogle Scholar
  74. 74.
    Pielage J, Stork T, Bunse I, Klämbt C (2003) The cell survival gene discs lost encodes a cytoplasmic Codanin-1 like protein, not a homolog of the tight junction PDZ-protein Patj. Dev Cell 5:841–851CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.National Institute of Technology Rourkela (NITR)RourkelaIndia
  2. 2.Max-Planck-Institute of Molecular Cell Biology and GeneticsDresdenGermany

Personalised recommendations