The Evolutionary Origination and Diversification of a Dimorphic Gene Regulatory Network through Parallel Innovations in and

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The genomic content of regulatory genes such as transcription factors is surprisingly conserved between diverse animal species, raising the paradox of how new traits emerge, and are subsequently modified and lost. In this study we make a connection between the developmental basis for the formation of a fruit fly trait and the evolutionary basis for that trait’s origin, diversification, and loss. We show how the origin of a novel pigmentation trait is associated with the evolution of two regulatory sequences that control the co-expression of two key pigmentation genes. These sequences interact in unique ways with evolutionarily conserved Hox transcription factors to drive gene co-expression. Once these unique connections evolved, the alteration of this trait appears to have proceeded through changes to regulatory genes rather than regulatory sequences of the pigmentation genes. Thus, our findings support a scenario where regulatory sequence evolution provided new functions to old transcription factors, how co-expression can emerge from different utilizations of the same transcription factors, and that trait diversity was surprisingly shaped by changes in some manner to the deeply conserved regulatory genes.

Vyšlo v časopise: The Evolutionary Origination and Diversification of a Dimorphic Gene Regulatory Network through Parallel Innovations in and. PLoS Genet 11(4): e32767. doi:10.1371/journal.pgen.1005136
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1005136

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Zdroje

1. Davidson EH, Erwin DH (2006) Gene regulatory networks and the evolution of animal body plans. Science 311 : 796–800. 16469913

2. Carroll SB (2008) Evo-devo and an expanding evolutionary synthesis: a genetic theory of morphological evolution. Cell 134 : 25–36. http://www.ncbi.nlm.nih.gov/pubmed/18614008. Accessed 24 July 2011. doi: 10.1016/j.cell.2008.06.030 18614008

3. Wray GA (2007) The evolutionary significance of cis-regulatory mutations. Nat Rev Genet 8 : 206–216. http://www.ncbi.nlm.nih.gov/pubmed/17304246. Accessed 17 July 2011. 17304246

4. Stern DL, Orgogozo V (2008) The loci of evolution: how predictable is genetic evolution? Evolution 62 : 2155–2177. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2613234&tool=pmcentrez&rendertype=abstract. Accessed 10 June 2011. doi: 10.1111/j.1558-5646.2008.00450.x 18616572

5. Martin A, Orgogozo V (2013) The Loci of repeated evolution: a catalog of genetic hotspots of phenotypic variation. Evolution 67 : 1235–1250. http://www.ncbi.nlm.nih.gov/pubmed/23617905. Accessed 6 August 2013. doi: 10.1111/evo.12081 23617905

6. Williams TM, Selegue JE, Werner T, Gompel N, Kopp A, et al. (2008) The regulation and evolution of a genetic switch controlling sexually dimorphic traits in Drosophila. Cell 134 : 610–623.http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2597198&tool=pmcentrez&rendertype=abstract. Accessed 4 August 2011. doi: 10.1016/j.cell.2008.06.052 18724934

7. Rebeiz M, Jikomes N, Kassner VA, Carroll SB (2011) Evolutionary origin of a novel gene expression pattern through co-option of the latent activities of existing regulatory sequences. Proc Natl Acad Sci U S A 108 : 10036–10043. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3121811&tool=pmcentrez&rendertype=abstract. Accessed 7 July 2011. doi: 10.1073/pnas.1105937108 21593416

8. Rebeiz M, Pool JE, Kassner VA, Aquadro CF, Carroll SB (2009) Stepwise modification of a modular enhancer underlies adaptation in a Drosophila population. Science 326 : 1663–1667. http://www.ncbi.nlm.nih.gov/pubmed/20019281. Accessed 2 August 2011. doi: 10.1126/science.1178357 20019281

9. Shirangi TR, Dufour HD, Williams TM, Carroll SB (2009) Rapid evolution of sex pheromone-producing enzyme expression in Drosophila. PLoS Biol 7: e1000168. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2711336&tool=pmcentrez&rendertype=abstract. Accessed 25 July 2011. doi: 10.1371/journal.pbio.1000168 19652700

10. Arnoult L, Su K, Manoel D, Minervino C, Magrina J, et al. (2013) Emergence and Diversification of Fly Pigmentation Through Evolution of a Gene Regulatory Module. Science 339 : 1423–1426. http://www.sciencemag.org/cgi/doi/10.1126/science.1233749. Accessed 22 March 2013. doi: 10.1126/science.1233749 23520110

11. Prud’homme B, Gompel N, Rokas A, Kassner VA, Williams TM, et al. (2006) Repeated morphological evolution through cis-regulatory changes in a pleiotropic gene. Nature 440 : 1050–1053. http://www.ncbi.nlm.nih.gov/pubmed/16625197. Accessed 10 July 2011. 16625197

12. Gompel N, Prud’homme B, Wittkopp PJ, Kassner VA, Carroll SB (2005) Chance caught on the wing: cis-regulatory evolution and the origin of pigment patterns in Drosophila. Nature 433 : 481–487. http://www.ncbi.nlm.nih.gov/pubmed/15690032. 15690032

13. Cretekos CJ, Wang Y, Green ED, Martin JF, Rasweiler JJ, et al. (2008) Regulatory divergence modifies limb length between mammals. Genes Dev 22 : 141–151. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2192750&tool=pmcentrez&rendertype=abstract. Accessed 21 July 2011. doi: 10.1101/gad.1620408 18198333

14. Prabhakar S, Visel A, Akiyama JA, Shoukry M, Lewis KD, et al. (2008) Human-specific gain of function in a developmental enhancer. Science 321 : 1346–1350. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2658639&tool=pmcentrez&rendertype=abstract. Accessed 30 July 2011. doi: 10.1126/science.1159974 18772437

15. Chan YF, Marks ME, Jones FC, Villarreal G, Shapiro MD, et al. (2010) Adaptive evolution of pelvic reduction in sticklebacks by recurrent deletion of a Pitx1 enhancer. Science 327 : 302–305. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3109066&tool=pmcentrez&rendertype=abstract. Accessed 30 July 2011. doi: 10.1126/science.1182213 20007865

16. Rogers WA, Salomone JR, Tacy DJ, Camino EM, Davis KA, et al. (2013) Recurrent Modification of a Conserved Cis-Regulatory Element Underlies Fruit Fly Pigmentation Diversity. PLoS Genet 9: e1003740. http://dx.plos.org/10.1371/journal.pgen.1003740. Accessed 1 September 2013. doi: 10.1371/journal.pgen.1003740 24009528

17. Spitz F, Gonzalez F, Duboule D (2003) A Global Control Region Defines a Chromosomal Regulatory Landscape Containing the HoxD Cluster. Cell 113 : 405–417. 12732147

18. Guerreiro I, Nunes A, Woltering JM, Casaca A, Nóvoa A, et al. (2013) Role of a polymorphism in a Hox/Pax-responsive enhancer in the evolution of the vertebrate spine. Proc Natl Acad Sci U S A 110 : 10682–10686. http://www.ncbi.nlm.nih.gov/pubmed/23674686. Accessed 14 November 2013. doi: 10.1073/pnas.1300592110 23674686

19. Frankel N, Erezyilmaz DF, McGregor AP, Wang S, Payre F, et al. (2011) Morphological evolution caused by many subtle-effect substitutions in regulatory DNA. Nature 474 : 598–603. http://www.nature.com/doifinder/10.1038/nature10200. Accessed 29 June 2011. doi: 10.1038/nature10200 21720363

20. Rebeiz M, Williams TM (2011) Experimental Approaches to Evaluate the Contributions of Candidate Cis -⁠ regulatory Mutations to Phenotypic Evolution. In: Orgogozo V, Rockman M V., editors. Methods in Molecular Biology. Methods in Molecular Biology. Totowa, NJ: Humana Press, Vol. 772. pp. 351–375. http://www.springerlink.com/index/10.1007/978-1-61779-228-1. Accessed 9 November 2011. doi: 10.1007/978-1-61779-228-1_21 22065449

21. Wittkopp PJ, Carroll SB, Kopp A (2003) Evolution in black and white: genetic control of pigment patterns in Drosophila. Trends Genet 19 : 495–504. http://linkinghub.elsevier.com/retrieve/pii/S016895250300194X. Accessed 17 July 2011. 12957543

22. Markow TA, O’Grady PM (2006) Drosophila: A guide to species identification and use. Academic Press.

23. Jeong S, Rokas A, Carroll SB (2006) Regulation of body pigmentation by the Abdominal-B Hox protein and its gain and loss in Drosophila evolution. Cell 125 : 1387–1399. http://www.ncbi.nlm.nih.gov/pubmed/16814723. Accessed 27 August 2011. 16814723

24. Kopp A, True JR (2002) Phylogeny of the Oriental Drosophila melanogaster species group: a multilocus reconstruction. Syst Biol 51 : 786–805. http://www.ncbi.nlm.nih.gov/pubmed/12396591. Accessed 19 November 2012. 12396591

25. Kopp A (2006) Basal relationships in the Drosophila melanogaster species group. Mol Phylogenet Evol 39 : 787–798. http://www.ncbi.nlm.nih.gov/pubmed/16527496. Accessed 21 April 2014. 16527496

26. Jeong S, Rebeiz M, Andolfatto P, Werner T, True J, et al. (2008) The evolution of gene regulation underlies a morphological difference between two Drosophila sister species. Cell 132 : 783–793. http://www.ncbi.nlm.nih.gov/pubmed/18329365. Accessed 10 June 2011. doi: 10.1016/j.cell.2008.01.014 18329365

27. Wittkopp PJ, Vaccaro K, Carroll SB (2002) Evolution of yellow gene regulation and pigmentation in Drosophila. Curr Biol 12 : 1547–1556. http://www.ncbi.nlm.nih.gov/pubmed/12372246. 12372246

28. True JR, Yeh S-D, Hovemann BT, Kemme T, Meinertzhagen IA, et al. (2005) Drosophila tan encodes a novel hydrolase required in pigmentation and vision. PLoS Genet 1: e63. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1285064&tool=pmcentrez&rendertype=abstract. Accessed 7 January 2012. 16299587

29. Wittkopp PJ, True JR, Carroll SB (2002) Reciprocal functions of the Drosophila yellow and ebony proteins in the development and evolution of pigment patterns. Development 129 : 1849–1858. http://www.ncbi.nlm.nih.gov/pubmed/11934851. 11934851

30. Ordway A, Hancuch KN, Johnson W, Wiliams TM, Rebeiz M (2014) The expansion of body coloration involves coordinated evolution in cis and trans within the pigmentation regulatory network of drosophila prostipennis. Dev Biol: 1–10. http://www.ncbi.nlm.nih.gov/pubmed/24907418. Accessed 10 June 2014.

31. Lachaise D, Harry M, Solignac M, Lemeunier F, Bénassi V, et al. (2000) Evolutionary novelties in islands: Drosophila santomea, a new melanogaster sister species from São Tomé. Proc Biol Sci 267 : 1487–1495. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1690712&tool=pmcentrez&rendertype=abstract. Accessed 6 March 2013. 11007323

32. Kopp A, Duncan I, Godt D, Carroll SB (2000) Genetic control and evolution of sexually dimorphic characters in Drosophila. Nature 408 : 553–559. doi: 10.1038/35046017 11117736

33. Salomone JR, Rogers WA, Rebeiz M, Williams TM (2013) The evolution of Bab paralog expression and abdominal pigmentation among Sophophora fruit fly species. Evol Dev 15 : 442–457. http://www.ncbi.nlm.nih.gov/pubmed/24261445. Accessed 24 November 2013. doi: 10.1111/ede.12053 24261445

34. Kalay G, Wittkopp PJ (2010) Nomadic enhancers: tissue-specific cis-regulatory elements of yellow have divergent genomic positions among Drosophila species. PLoS Genet 6: e1001222. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2996884&tool=pmcentrez&rendertype=abstract. Accessed 26 July 2012. doi: 10.1371/journal.pgen.1001222 21151964

35. Celniker SE, Lewis EB (1993) Molecular basis of transabdominal—a sexually dimorphic mutant of the bithorax complex of Drosophila. Proc Natl Acad Sci U S A 90 : 1566–1570. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=45915&tool=pmcentrez&rendertype=abstract. 8094560

36. Sánchez-Herrero E (1991) Control of the expression of the bithorax complex genes abdominal-A and abdominal-B by cis-regulatory regions in Drosophila embryos. Development 111 : 437–449. http://www.ncbi.nlm.nih.gov/pubmed/1680047. 1680047

37. Sanchez-Herrero E, Vernos I, Marco R, Morata G (1985) Genetic organization of Drosophila bithorax complex. Nature 313 : 108–113. 3917555

38. Kopp A, Duncan I (2002) Anteroposterior patterning in adult abdominal segments of Drosophila. Dev Biol 242 : 15–30. http://www.ncbi.nlm.nih.gov/pubmed/11795937. Accessed 19 February 2012. 11795937

39. Rogers WA, Grover S, Stringer SJ, Parks J, Rebeiz M, et al. (2014) A survey of the trans-regulatory landscape for Drosophila melanogaster abdominal pigmentation. Dev Biol 385 : 417–432. http://www.ncbi.nlm.nih.gov/pubmed/24269556. Accessed 18 December 2013. doi: 10.1016/j.ydbio.2013.11.013 24269556

40. Mann RS, Lelli KM, Joshi R (2009) Hox Specificity: Unique Roles for Cofactors and Collaborators. Curr Top Dev Biol 88 : 63–101. doi: 10.1016/S0070-2153(09)88003-4 19651302

41. Noyes MB, Christensen RG, Wakabayashi A, Stormo GD, Brodsky MH, et al. (2008) Analysis of homeodomain specificities allows the family-wide prediction of preferred recognition sites. Cell 133 : 1277–1289. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2478728&tool=pmcentrez&rendertype=abstract. Accessed 17 July 2012. doi: 10.1016/j.cell.2008.05.023 18585360

42. Carroll SB (1995) Homeotic genes and the evolution of arthropods and cordates. Nature 376 : 479–485. 7637779

43. Averof M, Patel NH (1997) Crustacean appendage evolution associated with changes in Hox gene expression. Nature 388 : 682–686. http://www.ncbi.nlm.nih.gov/pubmed/9262403. 9262403

44. Cohn MJ, Tickle C (1999) Developmental basis of limblessness and axial patterning in snakes. Nature 399 : 474–479. 10365960

45. Khila A, Abouheif E, Rowe L (2009) Evolution of a novel appendage ground plan in water striders is driven by changes in the Hox gene Ultrabithorax. PLoS Genet 5: e1000583. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2709915&tool=pmcentrez&rendertype=abstract. Accessed 26 July 2012. doi: 10.1371/journal.pgen.1000583 19649305

46. Weatherbee SD, Nijhout HF, Grunert LW, Halder G, Galant R, et al. (1999) Ultrabithorax function in butterfly wings and the evolution of insect wing patterns. Curr Biol 9 : 109–115. http://www.ncbi.nlm.nih.gov/pubmed/10021383. 10021383

47. Stansbury MS, Moczek AP (2014) The function of Hox and appendage-patterning genes in the development of an evolutionary novelty, the Photuris firefly lantern. Proc Biol Sci 281.

48. Tanaka K, Barmina O, Sanders LE, Arbeitman MN, Kopp A (2011) Evolution of sex-specific traits through changes in HOX-dependent doublesex expression. PLoS Biol 9: e1001131. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3160335&tool=pmcentrez&rendertype=abstract. Accessed 18 April 2014. doi: 10.1371/journal.pbio.1001131 21886483

49. Barmina O, Kopp A (2007) Sex-specific expression of a HOX gene associated with rapid morphological evolution. Dev Biol 311 : 277–286. http://www.ncbi.nlm.nih.gov/pubmed/17868668. Accessed 26 March 2014. 17868668

50. Walsh CM, Carroll SB (2007) Collaboration between Smads and a Hox protein in target gene repression. Development 134 : 3585–3592. http://www.ncbi.nlm.nih.gov/pubmed/17855427. Accessed 18 April 2014. 17855427

51. Gebelein B, McKay DJ, Mann RS (2004) Direct integration of Hox and segmentation gene inputs during Drosophila development. Nature 431 : 653–659. http://www.ncbi.nlm.nih.gov/pubmed/15470419. 15470419

52. Li-Kroeger D, Witt L, Grimes HL, Cook TA, Gebelein B (2008) Hox and senseless antagonism functions as a molecular switch to regulate EGF secretion in the Drosophila PNS. Dev Cell 15 : 298–308. http://www.ncbi.nlm.nih.gov/pubmed/20798606. Accessed 10 April 2012. doi: 10.1016/j.devcel.2008.06.001 18694568

53. Ryoo HD, Marty T, Casares F, Affolter M, Mann RS (1999) Regulation of Hox target genes by a DNA bound Homothorax/Hox/Extradenticle complex. Development 126 : 5137–5148. http://www.ncbi.nlm.nih.gov/pubmed/10529430. 10529430

54. Gebelein B, Culi J, Ryoo HD, Zhang W, Mann RS (2002) Specificity of Distalless repression and limb primordia development by abdominal Hox proteins. Dev Cell 3 : 487–498. http://www.ncbi.nlm.nih.gov/pubmed/12408801. 12408801

55. Stathopoulos A, Levine M (2005) Genomic regulatory networks and animal development. Dev Cell 9 : 449–462. http://www.ncbi.nlm.nih.gov/pubmed/16198288. Accessed 27 July 2011. 16198288

56. Bonn S, Furlong EEM (2008) cis-Regulatory networks during development: a view of Drosophila. Curr Opin Genet Dev 18 : 513–520. http://www.ncbi.nlm.nih.gov/pubmed/18929653. Accessed 14 March 2012. doi: 10.1016/j.gde.2008.09.005 18929653

57. Carroll SB, Grenier J, Weatherbee SD (2005) From DNA to Diversity: Molecular Genetics and the Evolution of Animal Design. 2nd ed. Malden, MA: Blackwell Publishing.

58. Kopp A, Graze RM, Xu S, Carroll SB, Nuzhdin S V (2003) Quantitative Trait Loci Responsible for Variation in Sexually Dimorphic Traits in Drosophila melanogaster. Genetics 787 : 771–787.

59. Bickel RD, Kopp A, Nuzhdin S V (2011) Composite effects of polymorphisms near multiple regulatory elements create a major-effect QTL. PLoS Genet 7: e1001275. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3020931&tool=pmcentrez&rendertype=abstract. Accessed 26 October 2012. doi: 10.1371/journal.pgen.1001275 21249179

60. Wittkopp PJ, Stewart EE, Arnold LL, Neidert AH, Haerum BK, et al. (2009) Intraspecific polymorphism to interspecific divergence: genetics of pigmentation in Drosophila. Science 326 : 540–544. http://www.ncbi.nlm.nih.gov/pubmed/19900891. Accessed 5 July 2011. doi: 10.1126/science.1176980 19900891

61. Werner T, Koshikawa S, Williams TM, Carroll SB (2010) Generation of a novel wing colour pattern by the Wingless morphogen. Nature 464 : 1143–1148. http://www.ncbi.nlm.nih.gov/pubmed/20376004. Accessed 15 July 2011. doi: 10.1038/nature08896 20376004

62. Rajakumar R, San Mauro D, Dijkstra MB, Huang MH, Wheeler DE, et al. (2012) Ancestral developmental potential facilitates parallel evolution in ants. Science 335 : 79–82. http://www.ncbi.nlm.nih.gov/pubmed/22223805. Accessed 13 August 2014. doi: 10.1126/science.1211451 22223805

63. Abouheif E (2008) Parallelism as the pattern and process of mesoevolution. Evol Dev 10 : 3–5. http://www.ncbi.nlm.nih.gov/pubmed/18184352. doi: 10.1111/j.1525-142X.2007.00208.x 18184352

64. Abouheif E, Fave M-J, Ibarraran-Viniegra AS, Lesoway MP, Rafiqi AM, et al. (2014) Eco-Evo-Devo: The Time Has Come. In: Landry CR, Aubin-Horth N, editors. Advances in Experimental Medicine and Biology. Springer. pp. 107–126. doi: 10.1007/978-3-319-06068-2_6 24952181

65. Erives A, Levine M (2004) Coordinate enhancers share common organizational features in the Drosophila genome. Proc Natl Acad Sci U S A 101 : 3851–3856. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=374333&tool=pmcentrez&rendertype=abstract. Accessed 30 November 2012. 15026577

66. Crocker J, Tamori Y, Erives A (2008) Evolution acts on enhancer organization to fine-tune gradient threshold readouts. PLoS Biol 6: e263. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2577699&tool=pmcentrez&rendertype=abstract. Accessed 1 March 2012. doi: 10.1371/journal.pbio.0060263 18986212

67. Markstein M, Pitsouli C, Villalta C, Celniker SE, Perrimon N (2008) Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes. Nat Genet 40 : 476–483. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2330261&tool=pmcentrez&rendertype=abstract. Accessed 17 July 2011. doi: 10.1038/ng.101 18311141

68. Bischof J, Maeda RK, Hediger M, Karch F, Basler K (2007) An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases. Proc Natl Acad Sci U S A 104 : 3312–3317. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1805588&tool=pmcentrez&rendertype=abstract. Accessed 21 June 2011. 17360644

69. Groth AC, Fish M, Nusse R, Calos MP (2004) Construction of Transgenic Drosophila by Using the Site-Specific Integrase From Phage phiC31. Genetics 166 : 1775–1782. 15126397

70. Calleja M, Herranz H, Estella C, Casal J, Lawrence P, et al. (2000) Generation of medial and lateral dorsal body domains by the pannier gene of Drosophila. Development 127 : 3971–3980. http://www.ncbi.nlm.nih.gov/pubmed/10952895. 10952895

71. Clark AG, Eisen MB, Smith DR, Bergman CM, Oliver B, et al. (2007) Evolution of genes and genomes on the Drosophila phylogeny. Nature 450 : 203–218. Available: http://www.ncbi.nlm.nih.gov/pubmed/17994087. Accessed 1 March 2012. 17994087

72. Richards S, Liu Y, Bettencourt BR, Hradecky P, Letovsky S, et al. (2005) Comparative genome sequencing of Drosophila pseudoobscura: chromosomal, gene, and cis-element evolution. Genome Res 15 : 1–18. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=540289&tool=pmcentrez&rendertype=abstract. Accessed 12 March 2012. 15632085

73. Frazer KA, Pachter L, Poliakov A, Rubin EM, Dubchak I (2004) VISTA: computational tools for comparative genomics. Nucleic Acids Res 32: W273–W279. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=441596&tool=pmcentrez&rendertype=abstract. Accessed 26 September 2013. 15215394

74. Rebeiz M, Posakony JW (2004) GenePalette: a universal software tool for genome sequence visualization and analysis. Dev Biol 271 : 431–438. 15223345

75. Rogers WA, Williams TM (2011) Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster. J Vis Exp: 2–7. http://www.ncbi.nlm.nih.gov/pubmed/22215325. Accessed 22 January 2012.

76. Barolo S, Castro B, Posakony JW (2004) New Drosophila transgenic reporters: insulated P-element vectors expressing fast-maturing RFP. Biotechniques 36 : 436–440, 442. http://www.ncbi.nlm.nih.gov/pubmed/15038159. 15038159

77. Abràmoff MD, Hospitals I, Magalhães PJ, Abràmoff M (2004) Image Processing with ImageJ. Biophotonics Int 11 : 36–42.