Identification of avoidance genes through neural pathway-specific forward optogenetics

English version

Autoři: Filipe Marques ^aff001; Gabriella Saro ^aff001; Andrei-Stefan Lia ^aff001; Richard J. Poole ^aff002; Laurent Falquet ^aff001; Dominique A. Glauser ^aff001
Působiště autorů: Department of Biology, University of Fribourg, Fribourg, Switzerland ^aff001; Department of Cell and Developmental Biology, University College London, London, United Kingdom ^aff002; Swiss Institute of Bioinformatics, Fribourg, Switzerland ^aff003
Vyšlo v časopise: Identification of avoidance genes through neural pathway-specific forward optogenetics. PLoS Genet 15(12): e32767. doi:10.1371/journal.pgen.1008509
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pgen.1008509

Souhrn

Understanding how the nervous system bridges sensation and behavior requires the elucidation of complex neural and molecular networks. Forward genetic approaches, such as screens conducted in C. elegans, have successfully identified genes required to process natural sensory stimuli. However, functional redundancy within the underlying neural circuits, which are often organized with multiple parallel neural pathways, limits our ability to identify ‘neural pathway-specific genes’, i.e. genes that are essential for the function of some, but not all of these redundant neural pathways. To overcome this limitation, we developed a ‘forward optogenetics’ screening strategy in which natural stimuli are initially replaced by the selective optogenetic activation of a specific neural pathway. We used this strategy to address the function of the polymodal FLP nociceptors mediating avoidance of noxious thermal and mechanical stimuli. According to our expectations, we identified both mutations in ‘general’ avoidance genes that broadly impact avoidance responses to a variety of natural noxious stimuli (unc-4, unc-83, and eat-4) and mutations that produce a narrower impact, more restricted to the FLP pathway (syd-2, unc-14 and unc-68). Through a detailed follow-up analysis, we further showed that the Ryanodine receptor UNC-68 acts cell-autonomously in FLP to adjust heat-evoked calcium signals and aversive behaviors. As a whole, our work (i) reveals the importance of properly regulated ER calcium release for FLP function, (ii) provides new entry points for new nociception research and (iii) demonstrates the utility of our forward optogenetic strategy, which can easily be transposed to analyze other neural pathways.

Klíčová slova:

Genetic screens – Genetically modified animals – Motor neurons – Nervous system – Neural pathways – Neurons – Optogenetics – Recombination reactions

Zdroje

1. Bargmann CI. Chemosensation in C. elegans. WormBook. 2006:1–29. Epub 2007/12/01. doi: 10.1895/wormbook.1.123.1 18050433.

2. Mori I. Genetics Of Chemotaxis And Thermotaxis In The Nematode Caenorhabditis Elegans. Annual Review of Genetics. 1999;33(1):399–422.

3. Schafer WR. Deciphering the Neural and Molecular Mechanisms of C. elegans Behavior. Current Biology. 2005;15(17):R723–R9. doi: 10.1016/j.cub.2005.08.020 16139205

4. Brenner S. Genetics of Caenorhabditis-Elegans. Genetics. 1974;77(1):71–94. WOS:A1974T582100007. 4366476

5. Konopka RJ, Benzer S. Clock mutants of Drosophila melanogaster. Proc Natl Acad Sci U S A. 1971;68(9):2112–6. Epub 1971/09/01. doi: 10.1073/pnas.68.9.2112 5002428; PubMed Central PMCID: PMC389363.

6. Ma X, Shen Y. Structural basis for degeneracy among thermosensory neurons in Caenorhabditis elegans. J Neurosci. 2012;32(1):1–3. Epub 2012/01/06. doi: 10.1523/JNEUROSCI.5112-11.2012 22219264.

7. Beverly M, Anbil S, Sengupta P. Degeneracy and neuromodulation among thermosensory neurons contribute to robust thermosensory behaviors in Caenorhabditis elegans. J Neurosci. 2011;31(32):11718–27. Epub 2011/08/13. doi: 10.1523/JNEUROSCI.1098-11.2011 21832201; PubMed Central PMCID: PMC3167209.

8. Trojanowski NF, Padovan-Merhar O, Raizen DM, Fang-Yen C. Neural and genetic degeneracy underlies Caenorhabditis elegans feeding behavior. J Neurophysiol. 2014;112(4):951–61. Epub 2014/05/30. doi: 10.1152/jn.00150.2014 24872529; PubMed Central PMCID: PMC4122747.

9. Ikeda M, Nakano S, Giles AC, Costa WS, Gottschalk A, Mori I. Circuit Degeneracy Facilitates Robustness and Flexibility of Navigation Behavior in C.elegans. BioRxiv. 2018. Epub 2018. https://doi.org/10.1101/385468

10. Mason PH. Degeneracy: Demystifying and destigmatizing a core concept in systems biology. Complexity. 2015;20(3):12–21. doi: 10.1002/cplx.21534

11. Chatzigeorgiou M, Schafer WR. Lateral facilitation between primary mechanosensory neurons controls nose touch perception in C. elegans. Neuron. 2011;70(2):299–309. Epub 2011/04/28. doi: 10.1016/j.neuron.2011.02.046 21521615; PubMed Central PMCID: PMC3145979.

12. Albeg A, Smith CJ, Chatzigeorgiou M, Feitelson DG, Hall DH, Schafer WR, et al. C. elegans multi-dendritic sensory neurons: morphology and function. Mol Cell Neurosci. 2011;46(1):308–17. Epub 2010/10/26. doi: 10.1016/j.mcn.2010.10.001 20971193; PubMed Central PMCID: PMC3018541.

13. Liu S, Schulze E, Baumeister R. Temperature- and touch-sensitive neurons couple CNG and TRPV channel activities to control heat avoidance in Caenorhabditis elegans. PLoS One. 2012;7(3):e32360. Epub 2012/03/27. doi: 10.1371/journal.pone.0032360 22448218; PubMed Central PMCID: PMC3308950.

14. Goodman MB, Sengupta P. How Caenorhabditis elegans Senses Mechanical Stress, Temperature, and Other Physical Stimuli. Genetics. 2019;212(1):25. doi: 10.1534/genetics.118.300241 31053616

15. Goodman MB. Mechanosensation. WormBook. 2006:1–14. Epub 2007/12/01. doi: 10.1895/wormbook.1.62.1 18050466; PubMed Central PMCID: PMC2806189.

16. Huang M, Chalfie M. Gene interactions affecting mechanosensory transduction in Caenorhabditis elegans. Nature. 1994;367(6462):467–70. Epub 1994/02/03. doi: 10.1038/367467a0 7509039.

17. Chatzigeorgiou M, Yoo S, Watson JD, Lee WH, Spencer WC, Kindt KS, et al. Specific roles for DEG/ENaC and TRP channels in touch and thermosensation in C. elegans nociceptors. Nat Neurosci. 2010;13(7):861–8. Epub 2010/06/01. doi: 10.1038/nn.2581 20512132; PubMed Central PMCID: PMC2975101.

18. Schild LC, Zbinden L, Bell HW, Yu YV, Sengupta P, Goodman MB, et al. The balance between cytoplasmic and nuclear CaM kinase-1 signaling controls the operating range of noxious heat avoidance. Neuron. 2014;84(5):983–96. Epub 2014/12/04. doi: 10.1016/j.neuron.2014.10.039 25467982; PubMed Central PMCID: PMC4318703.

19. Schild LC, Glauser DA. Dual Color Neural Activation and Behavior Control with Chrimson and CoChR in Caenorhabditis elegans. Genetics. 2015;200(4):1029–34. Epub 2015/05/30. doi: 10.1534/genetics.115.177956 26022242; PubMed Central PMCID: PMC4574232.

20. Edwards SL, Charlie NK, Milfort MC, Brown BS, Gravlin CN, Knecht JE, et al. A novel molecular solution for ultraviolet light detection in Caenorhabditis elegans. PLoS Biol. 2008;6(8):e198. Epub 2008/08/09. doi: 10.1371/journal.pbio.0060198 18687026; PubMed Central PMCID: PMC2494560.

21. Ward A, Liu J, Feng Z, Xu XZ. Light-sensitive neurons and channels mediate phototaxis in C. elegans. Nat Neurosci. 2008;11(8):916–22. Epub 2008/07/08. doi: 10.1038/nn.2155 18604203; PubMed Central PMCID: PMC2652401.

22. Liu J, Ward A, Gao J, Dong Y, Nishio N, Inada H, et al. C. elegans phototransduction requires a G protein-dependent cGMP pathway and a taste receptor homolog. Nat Neurosci. 2010;13(6):715–22. Epub 2010/05/04. doi: 10.1038/nn.2540 20436480; PubMed Central PMCID: PMC2882063.

23. Lee RY, Sawin ER, Chalfie M, Horvitz HR, Avery L. EAT-4, a homolog of a mammalian sodium-dependent inorganic phosphate cotransporter, is necessary for glutamatergic neurotransmission in caenorhabditis elegans. J Neurosci. 1999;19(1):159–67. Epub 1998/12/31. doi: 10.1523/JNEUROSCI.19-01-00159.1999 9870947; PubMed Central PMCID: PMC3759158.

24. Doitsidou M, Jarriault S, Poole RJ. Next-Generation Sequencing-Based Approaches for Mutation Mapping and Identification in Caenorhabditis elegans. Genetics. 2016;204(2):451–74. Epub 2016/10/13. doi: 10.1534/genetics.115.186197 27729495; PubMed Central PMCID: PMC5068839.

25. White JG, Southgate E, Thomson JN. Mutations in the Caenorhabditis elegans unc-4 gene alter the synaptic input to ventral cord motor neurons. Nature. 1992;355(6363):838–41. Epub 1992/02/27. doi: 10.1038/355838a0 1538764.

26. Sakamoto R, Byrd DT, Brown HM, Hisamoto N, Matsumoto K, Jin Y. The Caenorhabditis elegans UNC-14 RUN domain protein binds to the kinesin-1 and UNC-16 complex and regulates synaptic vesicle localization. Mol Biol Cell. 2005;16(2):483–96. doi: 10.1091/mbc.E04-07-0553 WOS:000226563600005. 15563606

27. Ogura K, Shirakawa M, Barnes TM, Hekimi S, Ohshima Y. The UNC-14 protein required for axonal elongation and guidance in Caenorhabditis elegans interacts with the serine/threonine kinase UNC-51. Gene Dev. 1997;11(14):1801–11. doi: 10.1101/gad.11.14.1801 WOS:A1997XM83500004. 9242488

28. Asakura T, Waga N, Ogura K, Goshima Y. Genes required for cellular UNC-6/netrin localization in Caenorhabditis elegans. Genetics. 2010;185(2):573–85. Epub 2010/04/13. doi: 10.1534/genetics.110.116293 20382828; PubMed Central PMCID: PMC2881138.

29. Lee RYN, Howe KL, Harris TW, Arnaboldi V, Cain S, Chan J, et al. WormBase 2017: molting into a new stage. Nucleic Acids Res. 2018;46(D1):D869–D74. Epub 2017/10/27. doi: 10.1093/nar/gkx998 29069413; PubMed Central PMCID: PMC5753391.

30. McGee MD, Rillo R, Anderson AS, Starr DA. UNC-83 is a KASH protein required for nuclear migration and is recruited to the outer nuclear membrane by a physical interaction with the SUN protein UNC-84. Mol Biol Cell. 2006;17(4):1790–801. doi: 10.1091/mbc.E05-09-0894 WOS:000236657900027. 16481402

31. Starr DA, Hermann GJ, Malone CJ, Fixsen W, Priess JR, Horvitz HR, et al. unc-83 encodes a novel component of the nuclear envelope and is essential for proper nuclear migration. Development. 2001;128(24):5039–50. WOS:000173434700010. 11748140

32. Horvitz HR, Sulston JE. Isolation and genetic characterization of cell-lineage mutants of the nematode Caenorhabditis elegans. Genetics. 1980;96(2):435–54. Epub 1980/10/01. 7262539; PubMed Central PMCID: PMC1214309.

33. Sulston JE, Horvitz HR. Abnormal cell lineages in mutants of the nematode Caenorhabditis elegans. Dev Biol. 1981;82(1):41–55. Epub 1981/02/01. doi: 10.1016/0012-1606(81)90427-9 7014288.

34. Zhen M, Jin Y. The liprin protein SYD-2 regulates the differentiation of presynaptic termini in C. elegans. Nature. 1999;401(6751):371–5. Epub 1999/10/12. doi: 10.1038/43886 10517634.

35. Goodwin PR, Juo P. The scaffolding protein SYD-2/Liprin-alpha regulates the mobility and polarized distribution of dense-core vesicles in C. elegans motor neurons. PLoS One. 2013;8(1):e54763. Epub 2013/01/30. doi: 10.1371/journal.pone.0054763 23358451; PubMed Central PMCID: PMC3554613.

36. Chia PH, Patel MR, Wagner OI, Klopfenstein DR, Shen K. Intramolecular regulation of presynaptic scaffold protein SYD-2/liprin-alpha. Mol Cell Neurosci. 2013;56:76–84. Epub 2013/04/02. doi: 10.1016/j.mcn.2013.03.004 23541703; PubMed Central PMCID: PMC3930023.

37. Wagner OI, Esposito A, Kohler B, Chen CW, Shen CP, Wu GH, et al. Synaptic scaffolding protein SYD-2 clusters and activates kinesin-3 UNC-104 in C. elegans. Proc Natl Acad Sci U S A. 2009;106(46):19605–10. Epub 2009/11/03. doi: 10.1073/pnas.0902949106 19880746; PubMed Central PMCID: PMC2780759.

38. Coronado R, Morrissette J, Sukhareva M, Vaughan DM. Structure and function of ryanodine receptors. Am J Physiol. 1994;266(6 Pt 1):C1485–504. doi: 10.1152/ajpcell.1994.266.6.C1485 8023884.

39. Sakube Y, Ando H, Kagawa H. An abnormal ketamine response in mutants defective in the ryanodine receptor gene ryr-1 (unc-68) of Caenorhabditis elegans. J Mol Biol. 1997;267(4):849–64. Epub 1997/04/11. doi: 10.1006/jmbi.1997.0910 9135117.

40. Maryon EB, Coronado R, Anderson P. unc-68 encodes a ryanodine receptor involved in regulating C. elegans body-wall muscle contraction. J Cell Biol. 1996;134(4):885–93. Epub 1996/08/01. doi: 10.1083/jcb.134.4.885 8769414; PubMed Central PMCID: PMC2120954.

41. Nagasaki K, Fleischer S. Ryanodine sensitivity of the calcium release channel of sarcoplasmic reticulum. Cell Calcium. 1988;9(1):1–7. Epub 1988/02/01. doi: 10.1016/0143-4160(88)90032-2 2452017.

42. Maryon EB, Saari B, Anderson P. Muscle-specific functions of ryanodine receptor channels in Caenorhabditis elegans. J Cell Sci. 1998;111 (Pt 19):2885–95. Epub 1998/09/10. 9730981.

43. Geffeney SL, Cueva JG, Glauser DA, Doll JC, Lee TH, Montoya M, et al. DEG/ENaC but not TRP channels are the major mechanoelectrical transduction channels in a C. elegans nociceptor. Neuron. 2011;71(5):845–57. Epub 2011/09/10. doi: 10.1016/j.neuron.2011.06.038 21903078; PubMed Central PMCID: PMC3170654.

44. Jose AM, Bany IA, Chase DL, Koelle MR. A specific subset of transient receptor potential vanilloid-type channel subunits in Caenorhabditis elegans endocrine cells function as mixed heteromers to promote neurotransmitter release. Genetics. 2007;175(1):93–105. Epub 2006/10/24. doi: 10.1534/genetics.106.065516 17057248; PubMed Central PMCID: PMC1774992.

45. Troemel ER, Kimmel BE, Bargmann CI. Reprogramming chemotaxis responses: sensory neurons define olfactory preferences in C. elegans. Cell. 1997;91(2):161–9. Epub 1997/11/05. doi: 10.1016/s0092-8674(00)80399-2 9346234.

46. Way JC, Chalfie M. The mec-3 gene of Caenorhabditis elegans requires its own product for maintained expression and is expressed in three neuronal cell types. Genes Dev. 1989;3(12A):1823–33. Epub 1989/12/01. doi: 10.1101/gad.3.12a.1823 2576011.

47. Liu Q, Chen B, Yankova M, Morest DK, Maryon E, Hand AR, et al. Presynaptic ryanodine receptors are required for normal quantal size at the Caenorhabditis elegans neuromuscular junction. J Neurosci. 2005;25(29):6745–54. Epub 2005/07/22. doi: 10.1523/JNEUROSCI.1730-05.2005 16033884.

48. Wei X, Potter CJ, Luo L, Shen K. Controlling gene expression with the Q repressible binary expression system in Caenorhabditis elegans. Nat Methods. 2012;9(4):391–5. Epub 2012/03/13. doi: 10.1038/nmeth.1929 22406855; PubMed Central PMCID: PMC3846601.

49. Fenno L, Yizhar O, Deisseroth K. The development and application of optogenetics. Annu Rev Neurosci. 2011;34:389–412. Epub 2011/06/23. doi: 10.1146/annurev-neuro-061010-113817 21692661.

50. Husson SJ, Costa WS, Wabnig S, Stirman JN, Watson JD, Spencer WC, et al. Optogenetic analysis of a nociceptor neuron and network reveals ion channels acting downstream of primary sensors. Curr Biol. 2012;22(9):743–52. Epub 2012/04/10. doi: 10.1016/j.cub.2012.02.066 22483941; PubMed Central PMCID: PMC3350619.

51. Wabnig S, Liewald JF, Yu SC, Gottschalk A. High-Throughput All-Optical Analysis of Synaptic Transmission and Synaptic Vesicle Recycling in Caenorhabditis elegans. PLoS One. 2015;10(8):e0135584. Epub 2015/08/28. doi: 10.1371/journal.pone.0135584 26312752; PubMed Central PMCID: PMC4552474.

52. Miller DM, Shen MM, Shamu CE, Burglin TR, Ruvkun G, Dubois ML, et al. C. elegans unc-4 gene encodes a homeodomain protein that determines the pattern of synaptic input to specific motor neurons. Nature. 1992;355(6363):841–5. Epub 1992/02/27. doi: 10.1038/355841a0 1347150.

53. McIntire SL, Garriga G, White J, Jacobson D, Horvitz HR. Genes necessary for directed axonal elongation or fasciculation in C. elegans. Neuron. 1992;8(2):307–22. Epub 1992/02/01. doi: 10.1016/0896-6273(92)90297-q 1739461.

54. Kittelmann M, Hegermann J, Goncharov A, Taru H, Ellisman MH, Richmond JE, et al. Liprin-alpha/SYD-2 determines the size of dense projections in presynaptic active zones in C. elegans. J Cell Biol. 2013;203(5):849–63. Epub 2013/12/11. doi: 10.1083/jcb.201302022 24322429; PubMed Central PMCID: PMC3857474.

55. Sun L, Shay J, McLoed M, Roodhouse K, Chung SH, Clark CM, et al. Neuronal regeneration in C. elegans requires subcellular calcium release by ryanodine receptor channels and can be enhanced by optogenetic stimulation. J Neurosci. 2014;34(48):15947–56. Epub 2014/11/28. doi: 10.1523/JNEUROSCI.4238-13.2014 25429136; PubMed Central PMCID: PMC4244466.

56. Sarasija S, Laboy JT, Ashkavand Z, Bonner J, Tang Y, Norman KR. Presenilin mutations deregulate mitochondrial Ca(2+) homeostasis and metabolic activity causing neurodegeneration in Caenorhabditis elegans. Elife. 2018;7. Epub 2018/07/11. doi: 10.7554/eLife.33052 29989545; PubMed Central PMCID: PMC6075864.

57. Busch KE, Laurent P, Soltesz Z, Murphy RJ, Faivre O, Hedwig B, et al. Tonic signaling from O(2) sensors sets neural circuit activity and behavioral state. Nat Neurosci. 2012;15(4):581–91. Epub 2012/03/06. doi: 10.1038/nn.3061 22388961; PubMed Central PMCID: PMC3564487.

58. Perez CG, Copello JA, Li Y, Karko KL, Gomez L, Ramos-Franco J, et al. Ryanodine receptor function in newborn rat heart. Am J Physiol Heart Circ Physiol. 2005;288(5):H2527–40. Epub 2005/01/01. doi: 10.1152/ajpheart.00188.2004 15626694.

59. Zalk R, Clarke OB, des Georges A, Grassucci RA, Reiken S, Mancia F, et al. Structure of a mammalian ryanodine receptor. Nature. 2015;517(7532):44–9. Epub 2014/12/04. doi: 10.1038/nature13950 25470061; PubMed Central PMCID: PMC4300236.

60. Euden J, Mason SA, Viero C, Thomas NL, Williams AJ. Investigations of the contribution of a putative glycine hinge to ryanodine receptor channel gating. J Biol Chem. 2013;288(23):16671–9. Epub 2013/05/02. doi: 10.1074/jbc.M113.465310 23632022; PubMed Central PMCID: PMC3675601.

61. Mei Y, Xu L, Mowrey DD, Mendez Giraldez R, Wang Y, Pasek DA, et al. Channel Gating Dependence on Pore Lining Helix Glycine Residues in Skeletal Muscle Ryanodine Receptor. J Biol Chem. 2015;290(28):17535–45. Epub 2015/05/23. doi: 10.1074/jbc.M115.659672 25998124; PubMed Central PMCID: PMC4498087.

62. Yemini E, Jucikas T, Grundy LJ, Brown AEX, Schafer WR. A database of Caenorhabditis elegans behavioral phenotypes. Nature Methods. 2013;10:877. doi: 10.1038/nmeth.2560 https://www.nature.com/articles/nmeth.2560#supplementary-information. 23852451

63. Javer A, Ripoll-Sánchez L, Brown André EX. Powerful and interpretable behavioural features for quantitative phenotyping of Caenorhabditis elegans. Philosophical Transactions of the Royal Society B: Biological Sciences. 2018;373(1758):20170375. doi: 10.1098/rstb.2017.0375 30201839

64. Stiernagle T. Maintenance of C. elegans. WormBook. 2006:1–11. Epub 2007/12/01. doi: 10.1895/wormbook.1.101.1 18050451; PubMed Central PMCID: PMC4781397.

65. Jorgensen EM, Mango SE. The art and design of genetic screens: caenorhabditis elegans. Nat Rev Genet. 2002;3(5):356–69. Epub 2002/05/04. doi: 10.1038/nrg794 11988761.

66. Shaham S. Counting mutagenized genomes and optimizing genetic screens in Caenorhabditis elegans. PLoS One. 2007;2(11):e1117. Epub 2007/11/09. doi: 10.1371/journal.pone.0001117 17989770; PubMed Central PMCID: PMC2065842.

67. Doitsidou M, Poole RJ, Sarin S, Bigelow H, Hobert O. C. elegans Mutant Identification with a One-Step Whole-Genome-Sequencing and SNP Mapping Strategy. Plos One. 2010;5(11). ARTN e15435 10.1371/journal.pone.0015435. WOS:000283920000025.

68. Minevich G, Park DS, Blankenberg D, Poole RJ, Hobert O. CloudMap: A Cloud-Based Pipeline for Analysis of Mutant Genome Sequences. Genetics. 2012;192(4):1249. doi: 10.1534/genetics.112.144204 23051646

69. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30(15):2114–20. doi: 10.1093/bioinformatics/btu170 24695404

70. Li H, Durbin R. Fast and accurate long-read alignment with Burrows–Wheeler transform. Bioinformatics. 2010;26(5):589–95. doi: 10.1093/bioinformatics/btp698 20080505

71. Kersey PJ, Allen JE, Allot A, Barba M, Boddu S, Bolt BJ, et al. Ensembl Genomes 2018: an integrated omics infrastructure for non-vertebrate species. Nucleic Acids Research. 2017;46(D1):D802–D8. doi: 10.1093/nar/gkx1011 29092050

72. Li H. A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data. Bioinformatics. 2011;27(21):2987–93. doi: 10.1093/bioinformatics/btr509 21903627

73. Van der Auwera GA, Carneiro MO, Hartl C, Poplin R, del Angel G, Levy-Moonshine A, et al. From FastQ Data to High-Confidence Variant Calls: The Genome Analysis Toolkit Best Practices Pipeline. Current Protocols in Bioinformatics. 2013;43(1):11.0.1–.0.33. doi: 10.1002/0471250953.bi1110s43 25431634

74. Poplin R, Ruano-Rubio V, DePristo MA, Fennell TJ, Carneiro MO, Van der Auwera GA, et al. Scaling accurate genetic variant discovery to tens of thousands of samples. bioRxiv. 2018:201178. doi: 10.1101/201178

75. Cingolani P, Platts A, Wang LL, Coon M, Nguyen T, Wang L, et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff. Fly. 2012;6(2):80–92. doi: 10.4161/fly.19695 22728672

76. Ruden D, Cingolani P, Patel V, Coon M, Nguyen T, Land S, et al. Using Drosophila melanogaster as a Model for Genotoxic Chemical Mutational Studies with a New Program, SnpSift. Frontiers in Genetics. 2012;3(35). doi: 10.3389/fgene.2012.00035 22435069

77. Li W, Kang L, Piggott BJ, Feng Z, Xu XZ. The neural circuits and sensory channels mediating harsh touch sensation in Caenorhabditis elegans. Nat Commun. 2011;2:315. Epub 2011/05/19. doi: 10.1038/ncomms1308 21587232; PubMed Central PMCID: PMC3098610.

78. Kaplan JM, Horvitz HR. A dual mechanosensory and chemosensory neuron in Caenorhabditis elegans. Proc Natl Acad Sci U S A. 1993;90(6):2227–31. Epub 1993/03/15. doi: 10.1073/pnas.90.6.2227 8460126; PubMed Central PMCID: PMC46059.

79. Hostettler L, Grundy L, Kaser-Pebernard S, Wicky C, Schafer WR, Glauser DA. The Bright Fluorescent Protein mNeonGreen Facilitates Protein Expression Analysis In Vivo. G3 (Bethesda). 2017;7(2):607–15. Epub 2017/01/22. doi: 10.1534/g3.116.038133 28108553; PubMed Central PMCID: PMC5295605.

80. Girard LR, Fiedler TJ, Harris TW, Carvalho F, Antoshechkin I, Han M, et al. WormBook: the online review of Caenorhabditis elegans biology. Nucleic Acids Res. 2007;35(Database issue):D472–5. Epub 2006/11/14. doi: 10.1093/nar/gkl894 17099225; PubMed Central PMCID: PMC1669767.

81. Evans TC. Transformation and microinjection. WormBook. 2006;10.

82. Tursun B, Cochella L, Carrera I, Hobert O. A toolkit and robust pipeline for the generation of fosmid-based reporter genes in C. elegans. PLoS One. 2009;4(3):e4625. Epub 2009/03/05. doi: 10.1371/journal.pone.0004625 19259264; PubMed Central PMCID: PMC2649505.