ART ic LE S Dissecting differential gene expression within the circadian neuronal circuit of Drosophila Emi Nagoshi1,2,4, Ken Sugino2, Ela Kula1,2,4, Etsuko Okazaki3, Taro Tachibana3, Sacha Nelson2 & Michael Rosbash1,2 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. A central goal of neurobiology is to understand animal behavior by the functional diversity of clock neurons and how they interact to understanding neuronal circuits. Deciphering how they function and form functional circuits remain largely unknown. thereby generate behavior requires identification of neuronal subtypes From a molecular standpoint, circadian rhythms are generated within a circuit, their organization and connections, as well as the by cell-autonomous molecular clocks present within many cells and contributions of individual cells to overall circuit output. Circadian tissues. In Drosophila, the core feedback loop consists of the trans- rhythms in Drosophila and the underlying brain neuronal circuit criptional activator CLOCK (CLK) and its partner CYCLE (CYC), provide an excellent substrate to achieve this end. Adult Drosophila which form a heterodimer and activate the transcription of period and locomotor activity peaks twice a day in anticipation of dawn and dusk timeless. The period (PER) and timeless (TIM) proteins then dimerize transitions, and approximately 150 clock-containing brain neurons and inhibit their own transcription by inactivating the CLK/CYC comprise the circuit controlling this circadian behavior. These clock complex. There is a subsidiary loop involving the transcription factor neurons are divided into seven classes on the basis of their anatomical VRILLE (VRI), which regulates Clock expression and thereby rein- locations and characteristics. There are three groups of dorsal neurons forces oscillations5. Many of these clock gene mRNAs manifest cir- © 2010 Nature America, Inc. All rights reserved. All rights Inc. America, Nature © 2010 (DN1, DN2 and DN3), lateral posterior neurons (LPN) and three cadian oscillations in abundance. Post-transcriptional modifications groups of lateral neurons: small ventral lateral neurons (s-LNv), large such as phosphorylation are also critical for rhythms and regulate the ventral lateral neurons (l-LNv) and dorsal lateral neurons (LNd). All stability and activity of clock transcription factors. The combination the l-LNvs and four out of five s-LNvs express the pigment-dispersing of transcriptional and post-transcriptional regulation ensures 24-h factor (PDF) neuropeptide. Larvae have a less complex yet still func- rhythmicity of the core clock. The core clock then regulates other tional circadian circuit1. Only three groups of circadian neurons, molecules, which accumulate rhythmically or have rhythmic activity, larval-LNv (lv-LNv), DN1 and DN2, are organized to effect larval to more directly generate overt rhythms of physiology or behavior6. circadian behaviors such as photoavoidance rhythms2. Many of these core clock or clock output molecules may not be Recent studies have shown that adult s-LNvs (morning oscilla- amenable to identification by forward genetic approaches, because of tor, M-cells) control morning activity and are essential for sustain- gene redundancy or additional essential functions during development. ing free-running rhythms in constant darkness, whereas a different For these reasons, many researchers have turned to microarray analysis group of cells, perhaps LNds and an s-LNv that does not express PDF of RNA from whole fly heads collected at different circadian times to (PDF-negative s-LNv, also known as 5th s-LNv), controls evening identify more candidate genes involved in the core clock or in circa- activity bouts and is thus defined as evening oscillator (E-cells)3,4. dian output. These studies have collectively identified many (100–200) Although this ‘M and E oscillator model’ seems to explain the basic rhythmically expressed (cycling) mRNAs in Drosophila heads7–12. principle of the circuit organization, the roles of many other clock However, surprisingly few new circadian-related genes have neurons remain undefined. Moreover, the molecules that underlie been discovered using the cycling gene expression in heads. 1Howard Hughes Medical Institute and 2National Center for Behavioral Genomics, Department of Biology, Brandeis University, Waltham, Massachusetts, USA. 3Department of Bioengineering, Graduate School of Engineering, Osaka City University, Osaka, Japan. 4Present addresses: Institute of Cell Biology, University of Bern, Bern, Switzerland (E.N.) and Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois, USA (E.K.). Correspondence should be addressed to M.R. ([email protected]). Received 25 June; accepted 19 October; published online 6 December 2009; doi:10.1038/nn.2451 60 VOLUME 13 | NUMBER 1 | JAnuARY 2010 NATURE NEUROSCIENCE ART ic LE S Figure 1 Gene expression profiling of clock cells a b Larva Adult in the Drosophila brain. (a) Strategy of the cell Dissociation of brains Fluorescently type–specific expression profiling. (b) GAL4 and elav-GAL4 labeled elav-GAL4 Isolation of GAL80 drivers used to label clock cells. Left, neurons labeled neurons expression of the drivers in the third-instar larval tim-GAL4 tim-GAL4 brain. Right, expression in the adult brain. DN1 DN2 RNA isolation DN1 DN2 5th s-LNv, also known as PDF-negative s-LNv. LNd DN3 5th s-LNv Pre-DN3 5th s-LNv (c) Microarray data for mRNAs enriched in clock Amplification and labeling Pdf-GAL4/ Pdf-GAL4/ Glia cells (see text). Except for head data, 0 to 2 Pdf-GAL80 Pdf-GAL80 Microarray Glia s-LNv indicate independent replicates. lv, larval; ad, LNv I-LNv adult; head, heads of wild-type flies collected at different time points. Head data series: 0 and 4 (independent replicates) are ZT3; 5 is Comparison c Reference ZT7; 1 and 6 are ZT11; 2 and 7 are ZT15; 8 is Pdf ZT 19; and 3 and 9 are ZT23. Left panel, data cry sets used for comparison and ranking. Middle tim tim columns, fold change (Fc) and t-test P-values. vri Hr51 Right panel, data sets used as reference. Top Fkbp13 Ir 40 genes (out of 84) that satisfied P < 0.001 CG13054 Side and Fc > 8 are shown (false discovery rate, MtnA Clk 1.42%). Scale bars: log2 of the signal level, log2 bnb of the fold change, and P-value –(log ). Arrows, 1631722_at 10 per probe sets for known clock genes; blue probe CG31324 Fer2 set names, bona fide pan-clock cell–enriched Thor CG31475 mRNAs, which are also more highly expressed CG7272 + – CG4726 in the lv-tim pdf cells and the eye than in elav cbt CG31705 cells; black probe set names, mRNAs that are 1624819_s_at not highly expressed in lv-tim+pdf– cells. 1638611_at 15.8 11.6 14.6 CG30282 CG17100 CG6749 CG8745 CG6310 One reason is that functionally important CG8557 /// DmirCG8557 CG32653 genes do not necessarily show cyclical RNA CG10508 CG5392 accumulation, as is the case in the cycle and Cyp12e1 Pdp1 13–15 CG33988 doubletime genes . The other possibil- ibug sfl ity is that mRNA cycling in only a small 1630614_s_at 2.04 3.13 3.06 2 0 0 1 1 2 3 4 5 6 7 8 9 0 1 2 number of clock neurons is masked by non- 0 1 2 0 1 2 0 1 0 1 2 0 1 2 0 1 2 Fc P Signal Fc P – log2 log2 –log10 Eye cycling mRNA in other neurons and head pdf + lv-tim Head lv-LNv lv-elav ad-elav ad l-LNv tissues, which makes cycling undetectable by ad s-LNv lv-tim microarray analysis. Furthermore, mRNAs that are only expressed in the clock circuit or a subset should be only a tiny fraction of head RNA and may RESULTS therefore escape detection in both cycling and noncycling analysis Gene expression profiling of Drosophila clock neurons © 2010 Nature America, Inc. All rights reserved. All rights Inc. America, Nature © 2010 of the head RNA. To profile gene expression of specific neurons within the Drosophila To further an understanding of circadian circuits and to circadian circuit, we adapted a method from previous gene expression circumvent mRNA oscillations as a defining property, we developed analyses of mouse brain neurons16,17. Briefly, targeted gene expres- a method to identify mRNAs enriched within different subsets of sion by the GAL4/UAS binary system and selective GAL80 expression well-defined types of brain neuron. Comparing the gene expres- was used to fluorescently label specific cell types with green fluores- sion profiles from different circadian cell groups, we identified cent protein (GFP) or yellow fluorescent protein (YFP). Dissected many new genes specifically expressed in all clock neurons or in brains were dissociated by mild protease treatment, and GFP- or important subclasses of clock neurons. We also characterized two of YFP-expressing single cells were collected manually. Total RNA was these new circadian genes, expressed specifically in subsets of clock isolated from approximately 100 cells, and poly(A) RNA was ampli- cells and affecting different aspects of rhythms. The previously fied and hybridized to an Affymetrix Drosophila genome expression uncharacterized transcription factor Fer2 is expressed in LNvs, and array (Fig.