Dissecting differential gene expression within the circadian neuronal circuit of Drosophila

Abstract

Behavioral circadian rhythms are controlled by a neuronal circuit consisting of diverse neuronal subgroups. To understand the molecular mechanisms underlying the roles of neuronal subgroups within the Drosophila circadian circuit, we used cell-type specific gene-expression profiling and identified a large number of genes specifically expressed in all clock neurons or in two important subgroups. Moreover, we identified and characterized two circadian genes, which are expressed specifically in subsets of clock cells and affect different aspects of rhythms. The transcription factor Fer2 is expressed in ventral lateral neurons; it is required for the specification of lateral neurons and therefore their ability to drive locomotor rhythms. The Drosophila melanogaster homolog of the vertebrate circadian gene nocturnin is expressed in a subset of dorsal neurons and mediates the circadian light response. The approach should also enable the molecular dissection of many different Drosophila neuronal circuits.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Gene expression profiling of clock cells in the Drosophila brain.
Figure 2: mRNAs enriched in the subclasses of circadian neurons.
Figure 3: Functional classifications of the mRNAs enriched in the clock neurons.
Figure 4: Fer2 expression is required for behavioral circadian rhythms.
Figure 5: The Fer2 gene is required for the specification of the lateral neurons.
Figure 6: nocturnin-RD is rhythmically expressed in a subset of clock neurons.
Figure 7: NOCTURNIN mediates light-mediated behavioral response in dorsal neurons.

Accession codes

Accessions

Gene Expression Omnibus

References

  1. 1

    Kaneko, M., Helfrich-Forster, C. & Hall, J.C. Spatial and temporal expression of the period and timeless genes in the developing nervous system of Drosophila: newly identified pacemakers candidates and novel features of clock gene product cycling. J. Neurosci. 17, 6745–6760 (1997).

    CAS  Article  Google Scholar 

  2. 2

    Mazzoni, E.O., Desplan, C. & Blau, J. Circadian pacemaker neurons transmit and modulate visual information to control a rapid behavioral response. Neuron 45, 293–300 (2005).

    CAS  Article  Google Scholar 

  3. 3

    Grima, B., Chelot, E., Xia, R. & Rouyer, F. Morning and evening peaks of activity rely on different clock neurons of the Drosophila brain. Nature 431, 869–873 (2004).

    CAS  Article  Google Scholar 

  4. 4

    Stoleru, D., Peng, Y., Agosto, J. & Rosbash, M. Coupled oscillators control morning and evening locomotor behaviour of Drosophila. Nature 431, 862–868 (2004).

    CAS  Article  Google Scholar 

  5. 5

    Yu, W. & Hardin, P.E. Circadian oscillators of Drosophila and mammals. J. Cell Sci. 119, 4793–4795 (2006).

    CAS  Article  Google Scholar 

  6. 6

    Gallego, M. & Virshup, D.M. Post-translational modifications regulate the ticking of the circadian clock. Nat. Rev. Mol. Cell Biol. 8, 139–148 (2007).

    CAS  Article  Google Scholar 

  7. 7

    Ceriani, M.F. et al. Genome-wide expression analysis in Drosophila reveals genes controlling circadian behavior. J. Neurosci. 22, 9305–9319 (2002).

    CAS  Article  Google Scholar 

  8. 8

    Claridge-Chang, A. et al. Circadian regulation of gene expression systems in the Drosophila head. Neuron 32, 657–671 (2001).

    CAS  Article  Google Scholar 

  9. 9

    Keegan, K.P., Pradhan, S., Wang, J.P. & Allada, R. Meta-analysis of Drosophila circadian microarray studies identifies a novel set of rhythmically expressed genes. PLoS Comput. Biol. 3, e208 (2007).

    Article  Google Scholar 

  10. 10

    Lin, Y. et al. Influence of the period-dependent circadian clock on diurnal, circadian, and aperiodic gene expression in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 99, 9562–9567 (2002).

    CAS  Article  Google Scholar 

  11. 11

    McDonald, M.J. & Rosbash, M. Microarray analysis and organization of circadian gene expression in Drosophila. Cell 107, 567–578 (2001).

    CAS  Article  Google Scholar 

  12. 12

    Ueda, H.R. et al. Genome-wide transcriptional orchestration of circadian rhythms in Drosophila. J. Biol. Chem. 277, 14048–14052 (2002).

    CAS  Article  Google Scholar 

  13. 13

    Park, J.H. et al. Differential regulation of circadian pacemaker output by separate clock genes in Drosophila. Proc. Natl. Acad. Sci. USA 97, 3608–3613 (2000).

    CAS  Article  Google Scholar 

  14. 14

    Rutila, J.E. et al. CYCLE is a second bHLH-PAS clock protein essential for circadian rhythmicity and transcription of Drosophila period and timeless. Cell 93, 805–814 (1998).

    CAS  Article  Google Scholar 

  15. 15

    Kloss, B. et al. The Drosophila clock gene double-time encodes a protein closely related to human casein kinase Iε. Cell 94, 97–107 (1998).

    CAS  Article  Google Scholar 

  16. 16

    Hempel, C.M., Sugino, K. & Nelson, S.B. A manual method for the purification of fluorescently labeled neurons from the mammalian brain. Nat. Protoc. 2, 2924–2929 (2007).

    CAS  Article  Google Scholar 

  17. 17

    Sugino, K. et al. Molecular taxonomy of major neuronal classes in the adult mouse forebrain. Nat. Neurosci. 9, 99–107 (2006).

    CAS  Article  Google Scholar 

  18. 18

    Kadener, S., Stoleru, D., McDonald, M., Nawathean, P. & Rosbash, M. Clockwork Orange is a transcriptional repressor and a new Drosophila circadian pacemaker component. Genes Dev. 21, 1675–1686 (2007).

    CAS  Article  Google Scholar 

  19. 19

    Murad, A., Emery-Le, M. & Emery, P. A subset of dorsal neurons modulates circadian behavior and light responses in Drosophila. Neuron 53, 689–701 (2007).

    CAS  Article  Google Scholar 

  20. 20

    Stoleru, D. et al. The Drosophila circadian network is a seasonal timer. Cell 129, 207–219 (2007).

    CAS  Article  Google Scholar 

  21. 21

    Moore, A.W., Barbel, S., Jan, L.Y. & Jan, Y.N. A genomewide survey of basic helix-loop-helix factors in Drosophila. Proc. Natl. Acad. Sci. USA 97, 10436–10441 (2000).

    CAS  Article  Google Scholar 

  22. 22

    Krapp, A. et al. The bHLH protein PTF1-p48 is essential for the formation of the exocrine and the correct spatial organization of the endocrine pancreas. Genes Dev. 12, 3752–3763 (1998).

    CAS  Article  Google Scholar 

  23. 23

    Hoshino, M. et al. Ptf1a, a bHLH transcriptional gene, defines GABAergic neuronal fates in cerebellum. Neuron 47, 201–213 (2005).

    CAS  Article  Google Scholar 

  24. 24

    Green, C.B. & Besharse, J.C. Identification of a novel vertebrate circadian clock-regulated gene encoding the protein nocturnin. Proc. Natl. Acad. Sci. USA 93, 14884–14888 (1996).

    CAS  Article  Google Scholar 

  25. 25

    Wang, Y. et al. Rhythmic expression of Nocturnin mRNA in multiple tissues of the mouse. BMC Dev. Biol. 1, 9 (2001).

    CAS  Article  Google Scholar 

  26. 26

    Garbarino-Pico, E. et al. Immediate early response of the circadian polyA ribonuclease nocturnin to two extracellular stimuli. RNA 13, 745–755 (2007).

    CAS  Article  Google Scholar 

  27. 27

    Green, C.B. et al. Loss of Nocturnin, a circadian deadenylase, confers resistance to hepatic steatosis and diet-induced obesity. Proc. Natl. Acad. Sci. USA 104, 9888–9893 (2007).

    CAS  Article  Google Scholar 

  28. 28

    Konopka, R.J., Pittendrigh, C. & Orr, D. Reciprocal behaviour associated with altered homeostasis and photosensitivity of Drosophila clock mutants. J. Neurogenet. 6, 1–10 (1989).

    CAS  Article  Google Scholar 

  29. 29

    Roy, P.J., Stuart, J.M., Lund, J. & Kim, S.K. Chromosomal clustering of muscle-expressed genes in Caenorhabditis elegans. Nature 418, 975–979 (2002).

    CAS  Article  Google Scholar 

  30. 30

    Doyle, J.P. et al. Application of a translational profiling approach for the comparative analysis of CNS cell types. Cell 135, 749–762 (2008).

    CAS  Article  Google Scholar 

  31. 31

    Heiman, M. et al. A translational profiling approach for the molecular characterization of CNS cell types. Cell 135, 738–748 (2008).

    CAS  Article  Google Scholar 

  32. 32

    Yang, Z., Edenberg, H.J. & Davis, R.L. Isolation of mRNA from specific tissues of Drosophila by mRNA tagging. Nucleic Acids Res. 33, e148 (2005).

    Article  Google Scholar 

  33. 33

    Hoopfer, E.D., Penton, A., Watts, R.J. & Luo, L. Genomic analysis of Drosophila neuronal remodeling: a role for the RNA-binding protein Boule as a negative regulator of axon pruning. J. Neurosci. 28, 6092–6103 (2008).

    CAS  Article  Google Scholar 

  34. 34

    Finkel, T. Oxygen radicals and signaling. Curr. Opin. Cell Biol. 10, 248–253 (1998).

    CAS  Article  Google Scholar 

  35. 35

    Lin, F.J., Song, W., Meyer-Bernstein, E., Naidoo, N. & Sehgal, A. Photic signaling by cryptochrome in the Drosophila circadian system. Mol. Cell. Biol. 21, 7287–7294 (2001).

    CAS  Article  Google Scholar 

  36. 36

    Sathyanarayanan, S. et al. Identification of novel genes involved in light-dependent CRY degradation through a genome-wide RNAi screen. Genes Dev. 22, 1522–1533 (2008).

    CAS  Article  Google Scholar 

  37. 37

    Rieger, D., Shafer, O.T., Tomioka, K. & Helfrich-Forster, C. Functional analysis of circadian pacemaker neurons in Drosophila melanogaster. J. Neurosci. 26, 2531–2543 (2006).

    CAS  Article  Google Scholar 

  38. 38

    Picot, M., Cusumano, P., Klarsfeld, A., Ueda, R. & Rouyer, F. Light activates output from evening neurons and inhibits output from morning neurons in the Drosophila circadian clock. PLoS Biol. 5, e315 (2007).

    Article  Google Scholar 

  39. 39

    Bahn, J.H., Lee, G. & Park, J.H. Comparative analysis of Pdf-mediated circadian behaviors between Drosophila melanogaster and D. virilis. Genetics 181, 965–975 (2009).

    CAS  Article  Google Scholar 

  40. 40

    Gronke, S., Bickmeyer, I., Wunderlich, R., Jackle, H. & Kuhnlein, R.P. curled encodes the Drosophila homolog of the vertebrate circadian deadenylase Nocturnin. Genetics 183, 219–232 (2009).

    Article  Google Scholar 

  41. 41

    Emery, P. et al. Drosophila CRY is a deep brain circadian photoreceptor. Neuron 26, 493–504 (2000).

    CAS  Article  Google Scholar 

  42. 42

    Yoshii, T., Todo, T., Wulbeck, C., Stanewsky, R. & Helfrich-Forster, C. Cryptochrome is present in the compound eyes and a subset of Drosophila's clock neurons. J. Comp. Neurol. 508, 952–966 (2008).

    CAS  Article  Google Scholar 

  43. 43

    Jiang, S.A., Campusano, J.M., Su, H. & O'Dowd, D.K. Drosophila mushroom body Kenyon cells generate spontaneous calcium transients mediated by PLTX-sensitive calcium channels. J. Neurophysiol. 94, 491–500 (2005).

    CAS  Article  Google Scholar 

  44. 44

    Kuppers-Munther, B. et al. A new culturing strategy optimises Drosophila primary cell cultures for structural and functional analyses. Dev. Biol. 269, 459–478 (2004).

    Article  Google Scholar 

  45. 45

    Tusher, V.G., Tibshirani, R. & Chu, G. Significance analysis of microarrays applied to the ionizing radiation response. Proc. Natl. Acad. Sci. USA 98, 5116–5121 (2001).

    CAS  Article  Google Scholar 

  46. 46

    Kaneko, M. & Hall, J.C. Neuroanatomy of cells expressing clock genes in Drosophila: transgenic manipulation of the period and timeless genes to mark the perikarya of circadian pacemaker neurons and their projections. J. Comp. Neurol. 422, 66–94 (2000).

    CAS  Article  Google Scholar 

  47. 47

    Klarsfeld, A. et al. Novel features of cryptochrome-mediated photoreception in the brain circadian clock of Drosophila. J. Neurosci. 24, 1468–1477 (2004).

    CAS  Article  Google Scholar 

  48. 48

    Kishiro, Y., Kagawa, M., Naito, I. & Sado, Y. A novel method of preparing rat-monoclonal antibody-producing hybridomas by using rat medial iliac lymph node cells. Cell Struct. Funct. 20, 151–156 (1995).

    CAS  Article  Google Scholar 

  49. 49

    Shafer, O.T., Rosbash, M. & Truman, J.W. Sequential nuclear accumulation of the clock proteins period and timeless in the pacemaker neurons of Drosophila melanogaster. J. Neurosci. 22, 5946–5954 (2002).

    CAS  Article  Google Scholar 

  50. 50

    Houl, J.H., Ng, F., Taylor, P. & Hardin, P.E. CLOCK expression identifies developing circadian oscillator neurons in the brains of Drosophila embryos. BMC Neurosci. 9, 119 (2008).

    Article  Google Scholar 

Download references

Acknowledgements

We thank P. Hardin (Texas A&M University) for antisera to CLK as well as the Bloomington Stock Center, National Institute of Genetics of Japan (NIG) and Vienna Drosophila RNAi Center (VDRC) for fly stocks. We are grateful to J. Menet, W. Luo, K. Abruzzi, S. Bradley, L. Griffith, P. Garrity, S. Waddell, R. Allada, Y. Shang, P. Emery and J. Blau for comments on the manuscript. Y. Shang also provided the protocol for culture media preparation, and N. Francis helped with brain dissections. Some of this work was supported by a fellowship to E.N. from the Charles A. King Trust.

Author information

Affiliations

Authors

Contributions

E.N. and M.R. conceived the idea of this project and wrote the manuscript; E.N. conducted molecular and behavioral experiments; K.S. conducted the microarray data analysis; E.K. performed the expression profiling of the adult neurons; K.S. and S.N. contributed to the development of the protocol for the Drosophila cell type–specific expression profiling; E.O. and T.T. generated a NOC-RD specific antibody.

Corresponding author

Correspondence to Michael Rosbash.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–4 and Supplementary Figures 1–4 (PDF 4836 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Nagoshi, E., Sugino, K., Kula, E. et al. Dissecting differential gene expression within the circadian neuronal circuit of Drosophila. Nat Neurosci 13, 60–68 (2010). https://doi.org/10.1038/nn.2451

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing