Neuroanatomy of cells expressing clock genes inDrosophila: Transgenic manipulation of theperiod andtimeless genes to mark the perikarya of circadian pacemaker neurons and their projections

Neuroanatomy of Cells Expressing ClockGenes in Drosophila: Transgenic

Manipulation of the period and timelessGenes to Mark the Perikarya ofCircadian Pacemaker Neurons

and their Projections

MAKI KANEKO AND JEFFREY C. HALL*

Department of Biology, Brandeis University, Waltham, Massachusetts 02454

ABSTRACTSubsets of brain neurons expressing the clock genes period (per) and timeless (tim)

are involved in the generation of circadian behavioral rhythms. However, current knowl-edge of projection patterns of these neurons is limited to those immunoreactive to anantibody against a crustacean neuropeptide. The GAL4-expression system was utilized tovisualize neuronal processes from all per and tim-expressing neurons in the centralnervous system. Each of two types of GAL4-driver fusion genes, per-gal4 or tim-gal4, wascombined in transgenic flies with marker genes—lacZ, and sequences encoding greenfluorescent protein or TAU protein— under the control of the GAL4-responsive elementUAS. This allowed visualization of the cytoplasm of GAL4-expressing cells. Thus, neu-rites of clock neurons in the adult brain as well as those of larvae and pupae wererevealed. Among the anatomical patterns revealed by per-gal4- or tim-gal4-driven markerexpression were a previously unknown, dorsally located neuronal cluster, along with theprojections of these cells and of other dorsal neurons characterized in earlier studies onlyby the location of their perikarya. The similarity of projections from PER- or TIM-containing neurons during development to those in the adult implies that these featuresof mature clock neurons are established by the larval stages. Neurons that have neverbeen identified as PER- or TIM-immunoreactive were also visualized in this assay system,indicating promoter activity of the clock genes in these cells and suggesting that theirproducts cannot accumulate to detectable levels in certain neurons. J. Comp. Neurol. 422:66 –94, 2000. © 2000 Wiley-Liss, Inc.

Indexing terms: circadian rhythms; developing and adult insect brains; per-gal4 and tim-gal4

fusion genes; b-galactosidase, TAU, and GFP marking; rhythm mutants

Circadian pacemakers are associated with specific ana-tomical structures in a variety of animals. Examples in-clude the basal retinal neurons of the sea slug (Whitmoreand Block, 1996), the optic lobe of the cockroach (Helfrich-Forster et al., 1998), and the suprachiasmatic nucleus inthe mammalian brain (Weaver, 1998). In Drosophila, theisolation of rhythm mutants and their correspondinggenes came before the identification of cells and tissuesthat possess clock functions (reviewed by Kaneko, 1998).The molecular-genetic studies led to identification of nu-merous putative clock cells throughout the fly’s body (re-viewed by Hall, 1995).

Although the biological function of most of these clockgene-expressing cells has not been defined, earlier studies

Grant Sponsor: National Institute of Health; Grant number: GM-33205.Dr. Kaneko’s current address is: Department of Biology and Biochemis-

try, University of Houston, Houston, TX 77204.*Correspondence to: Jeffrey C. Hall, Department of Biology, Brandeis

University, 415 South Street, Waltham, MA 02454-9110.E-mail: [email protected]

Received 12 October 1999; Revised 19 January 2000; Accepted 10 Feb-ruary 2000

THE JOURNAL OF COMPARATIVE NEUROLOGY 422:66–94 (2000)

© 2000 WILEY-LISS, INC.

involving brain transplants and genetic mosaic analysisestablished that a circadian pacemaker for behavioral ac-tivity resides in the brain of the adult fly (Handler andKonopka, 1979; Konopka et al., 1983; Ewer et al., 1992).The period gene (per) is expressed in discrete clusters ofneurons and in many glial cells within the brain (reviewedby Helfrich-Forster, 1996; Helfrich-Forster et al., 1998;Kaneko, 1998). Among these cells, so-called lateral neu-rons (LNs) located between the central brain and the opticlobes were shown to be necessary and sufficient for thegeneration of robust adult behavioral rhythms (Frisch etal., 1994; Helfrich-Forster, 1998), although per expressionin some glial cells may be sufficient for weak rhythmicity(Ewer et al., 1992).

Lateral neuronal cells that contain per gene productsare divided into three clusters (Helfrich-Forster, 1996;Kaneko, 1998): five to six neurons located relatively dor-sally (LNds); four to five more ventrally located neuronswith large somata (large LNvs); and four to five ventrolat-eral neurons with relatively small somata (small LNvs).Within certain of these clusters, four large LNvs and foursmall LNvs are immunostained by an antibody againstcrustacean pigment dispersing hormone (PDH; Helfrich-Forster and Homberg, 1993; Helfrich-Forster, 1995). His-tological and behavioral studies of mutants have revealedthe importance of the small LNvs for circadian locomotorrhythms. Most individual flies expressing a disconnected(disco) mutation are arrhythmic (Dushay et al., 1989; Har-din et al., 1992) and are devoid of LNds and LNvs (Zerr etal., 1990). The behavioral rhythmicity exhibited by a smallfraction of disco individuals is correlated with the pres-ence of small LNvs and their dorsally projecting neurites(Helfrich-Forster, 1998). The cryptochrome mutant cryb

lacks protein encoded by per (PER) and protein encoded bytim (TIM) cycling in most tissues, but cryb flies are behav-iorally rhythmic; this is correlated with periodic fluctua-tions of PER and TIM specifically in the small LNv cells(Stanewsky et al., 1998).

Although these studies indicate that the small LNvsmay be the most important pacemaker cells underlyinglocomotor activity rhythms, the other lateral-neuronalclusters and those of dorsally located neurons that express

per and tim (Kaneko, 1998) may also play important rolesin terms of controlling rhythmic behavior. In this regard,LN-less disco flies still entrain to light:dark (LD) cycles(Dushay et al., 1989; Hardin et al., 1992), and some ofthem remain rhythmic in constant darkness for a few days(Wheeler et al., 1993; Helfrich-Forster, 1998). Also, flies inwhich the PDH-immunoreactive LNvs were geneticallyablated are defective in circadian locomotor rhythms, butthey are not as severely arrhythmic as are the disco mu-tants (Renn et al., 1999).

Compared to PER expression patterns, cells and tissuesthat express the timeless gene have not been analyzedextensively. However, PER and TIM were found to becoexpressed in neuronal clusters in the larval brain(Kaneko et al., 1997). In adults, TIM immunoreactivityhas been detected in several tissues and cells that alsoexpress PER: photoreceptors in the retina, certain neu-rons, and glia in the brain, and Malpighian tubules(Hunter-Ensor et al., 1996; Giebultowicz and Hege, 1997;Stanewsky et al., 1998).

To gain a thorough understanding of how clock genesfunction in circadian pacemaker neurons to generaterhythmic outputs, the pathways from cells expressingclock genes must be determined. However, this informa-tion is available only for the PDH-immunoreactive LNvs(Helfrich-Forster and Homberg, 1993; Helfrich-Forster,1995), because PER and TIM as well as PER-b-galactosidase (bGAL) fusion proteins are predominantlylocalized in the cell bodies (Siwicki et al., 1988; Liu et al.,1992; Stanewsky et al., 1997a). In order to visualize per- ortim-expressing neuronal processes, we applied the binaryGAL4 transgene system, which has only begun to be ap-plied to histological analysis of the fly’s rhythm system(Plautz et al., 1997a). Thus, we used transgenes in whichregulatory regions of per and tim were fused to GAL4-encoding sequences. Expression of secondary reporter fac-tors under the control of these GAL4 drivers should markonly the cells expressing per or tim. As marker genes, weused three types of fusion constructs, each including theGAL4-responsive UAS sequences: lacZ, which encodes theE. coli b-galactosidase; green fluorescent protein (gfp),which encodes green fluorescent protein (GFP), a jellyfish

Abbreviations

AI antennal lobe relay interneuronAL antennal lobebGAL b-galactosidaseBG transgene encoding part of PER fused to bGALCA corpus allatumCC corpus cardiacumClkJrk Clock mutantcyc01 cycle mutantdbt double-time mutantdisco disconnected mutantDN dorsal neuronDN1 dorsal neuron, group 1DN2 dorsal neuron, group 2DN3 dorsal neuron, group 3DN1L larval dorsal neuron, group 1DN2L larval dorsal neuron, group 2DGI dorsal giant interneuronEB ellipsoid bodyELAV protein encoded by the pan-neural elav geneFB fan-shaped bodygal4 yeast gene encoding GAL4 transcription factorGFP green fluorescent protein

H-B Hofbauer-Buchner eyeletL3 third-instar larvaLD light:dark cycleLN lateral neuronLNv ventral lateral neuronLNd dorsal lateral neuronLTr lateral trianglemACT middle antenno-cerebral tractpdf pigment-dispersing factor genePDH Pigment Dispersing Hormoneper period genePER protein encoded by perPI pars intercerebralisPOT posterior optic tractSEG subesophageal gangliontim timeless geneTIM protein encoded by timTRI tritocerebrumUAS GAL4-responsive upstream activating sequencesWT wild-typeZT Zeitgeber time in LD cycle

67CLOCK-GENE-EXPRESSING NEURONS IN DROSOPHILA

protein used to fill axons in transgenic animals (e.g.,Chalfie et al., 1994); and tau, which encodes TAU, amicrotubule-associated protein distributed throughoutthe cytoplasm of neurons (e.g., Tucker, 1990). In a givendoubly transgenic type, carrying, for example, per-gal4along with UAS-lacZ, we analyzed marker expression dur-ing postembryonic development and in adults.

The principal aims were to reveal the projections ofclock gene-expressing cells previously recognized only asperikarya; to compare the patterns of cells in which theperiod and timeless genes are active; and to determinewhether stainings mediated by anti-PER and anti-TIMreveal the complete pattern of brain cells in which theseclock genes are transcribed. Furthermore, we testedmarker patterns in adults and larvae expressing the cy-cle01 (cyc01) and ClockJrk (ClkJrk) clock mutations. In thesemutants, the levels of per and tim products are muchlower than normal in tissue extracts (Allada et al., 1998;Rutila et al., 1998), but there are no data from in situanalysis of cyc01 and ClkJrk. Thus, it is not known whetherthese mutations have uniform effects on per- and tim-expressing cells.

MATERIALS AND METHODS

Rearing conditions

Strains of Drosophila melanogaster were reared in amedium containing agar, yeast, corn meal, dextrose, andthe mold-inhibitor Lexgard. In most cases, flies wereraised and otherwise maintained in 12-hour:12-hour LDcycle, at 25°C. Some rearings occurred in quasi-naturalday-night conditions of light and temperature fluctuationsin a laboratory room. In these cases, flies were transferredto controlled conditions of 12-hour:12-hour LD cycles at25°C, 3 days before they were histologically processed.

Fly stocks

GAL4-driver strains. Two such transgenic types wereused: per-promoter-gal4 and tim-promoter-gal4 (“promoter”refers to 59-flanking regions containing gene regulatorysequences in addition to the promoters per se). The originsof these molecular constructs and generations of thetransgenic strains were documented previously (Plautz etal., 1997a; Emery et al., 1998). One GAL4-driver line ofper-gal4, in which the transgene is inserted in chromo-some 2, was initially generated. Five additional transgeniclines of this type were generated by standard crossesaimed at mobilizing per-gal4. All of the further lines hadthe transgene inserted in chromosome 2. Twenty-threeindependent tim-gal4 driver lines were generated, 12 ofwhich were on chromosome 2, 10 on chromosome 3, andone on the X chromosome.

UAS-marker strains. For visualization of GAL4-expressing cells, the driver strains were crossed to trans-genic flies containing UAS-lacZ (Brand and Perrimon,1993), or UAS-gfpS65T (Ito et al., 1997a), or UAS-tau (Itoet al., 1997a, b). None of these marker-gene productscontains a nuclear localization signal. One line each wasused for UAS-lacZ (Bg4-2-4b) and UAS-gfpS65T (T2). TwoUAS-tau lines (24/1 and 7) were employed.

Strains containing both GAL4-driver and UAS-marker

transgenes. To facilitate repeated observations of fliesexpressing TAU or GFP under the control of per-gal4 ortim-gal4, the following genetically stable stocks were gen-

erated, each containing two types of transgenes: (1) per-gal4 (line 2)/CyO; UAS-tau (line 24/1, homozygous; CyO isa marked, multiply inverted second-chromosome balanc-er); (2) tim-gal4 (62, 82, or 86) UAS-tau (7)/CyO; (3) per-gal4 (1b, 2, or 3) UAS-gfp/CyO; (4) tim-gal4 (27, 62, 82, or86) UAS-gfp/CyO. Among the lines carrying UAS-gfp andper-gal4 or tim-gal4, only the following strains could havethe transgene-carrying chromosome 2 rendered homozy-gous (i.e., in viable adults): those carrying tim-gal4 (62),per-gal4 (1b), or per-gal4 (3) in addition to UAS-gfp. Es-sentially the same expression patterns of GFP were foundin these genetically stable stocks compared to the patternsin the progeny resulting from matings of two separatetransgenic types (e.g., per-gal4 3 UAS-gfp).

Additional strains. In addition to the GAL4-driverand UAS-marker lines listed above, a strain carrying thewhite eye-color mutation (without any transgenes) wasused for anti-TIM staining of sections. Two lines carryinga per-lacZ fusion gene (encoding two-thirds of PER fusedto bGAL) were used for double-labeling experiments in-volving anti-bGAL and anti-protein encoded by the pan-

TABLE 1. Strategies and Methods for Histological Detection of Clock Geneand Marker Expressions1

Strain2 Primary antibody(dilution)

Methods

UAS-lacZ Mouse monoclonal anti-bGAL(Promega, Madison, WI) [1:2,000]

Horizontal sections; peroxidase-mediated stain3 (Stanewsky etal., 1997a)

UAS-tau Bovine anti-TAU2 (Sigma, St.Louis, MO) [1:2000]

Sections; peroxidase3 (Stanewskyet al., 1997a)

UAS-tau Bovine anti-TAU2 (Sigma) [1:2000]

Wholemounts;immunofluorescence4 (Kaneko etal., 1997)

UAS-tau Anti-TAU (as above) and ratpolyclonal anti-TIM (1:4,000)

Wholemounts; immunoflourescentdouble-labeling5 (Kaneko et al.,1997)

per-lacZ (BG) Rabbit polyclonal anti-bGAL(Organon-Teknika; 1:1,500)and mouse monoclonal anti-ELAV (O’Neill et al., 1994;1:10 or 1:2)

Sections; immunoflourescentdouble-labeling6 (cf. Ewer et al.,1992)

per-lacZ (BG) Anti-bGAL and anti-ELAV (asabove; anti-ELAV 1:10)

Wholemounts; immunoflourescentdouble-labeling6 (Kaneko et al.,1997)

white Anti-TIM (as above) Sections; peroxidase3 (Stanewskyet al., 1997a)

UAS-gfp None Wholemounts; GFP-mediatedfluorescence7 (Ito et al., 1997a)

1 For abbreviations, see list (for this and subsequent tables).2 All strains except white are transgenics. The UAS-containing transgenes had theirmarkers expressed under the control of per-gal4 or tim-gal4 in experimental prepara-tions or were present alone in controls performed to assess background signals.3 Stained sections were observed by using Nomarski optics attached to a Zeiss Axiophotor an Axioskop microscope.4 Anti-TAU of brains from larvae and adults used a biotinylated secondary antibody(Jackson Immunoresearch, West Grove, PA) and streptavidin-fluorescein isothiocya-nate (Jackson Immunoresearch), except that adult specimens were fixed in 4% para-formaldehyde, phosphate-buffered (pH 7.4), for 4 hours at 10°C before the brains weredissected. Immunofluorescently labeled wholemounted brains were observed with anMRC600 laser-scanning confocal microscope (BioRad, Richmond, CA).5 Specimens were fixed as described above. For these double-labelings, no cross-reactivities between anti-rat IgG and mouse anti-TAU antibody or between anti-mouseIgG and rat anti-TIM antiserum were observed.6 Anti-ELAV exclusively marks all Drosophila neurons (Yao et al., 1993). Thus, thesedouble-labelings of adult brains were performed to identify the neuronal vs. glial natureof cells within a newly identified per-expressing dorsal group. For the sections, threeflies from the homozygous BG6a line were killed at ZT 0. For the wholemounts, fourflies from homozygous BG6a line and two from BG/TM6B Tb were killed at ZT23(brains dissected from flies fixed as above). Fluorescent dye-tagged secondary antibod-ies were used, and the specimens were observed with the confocal microscope. Nocross-reactivities between anti-mouse IgG and rabbit anti-bGAL or between anti-rabbitIgG and anti-ELAV (mAb 5D) were detected.7 GFP images of whole adults and larvae, along with their body parts and internalorgans, were observed under a Zeiss Axiophot microscope or a Leica dissecting micro-scope, both equipped with epifluorescent light sources and GFP filters. Fluorescencemediated by GFP in wholemounted brains was observed with the confocal microscope.

68 M. KANEKO AND J.C. HALL

neural elav gene (ELAV, Table 1); this transgenic type iscalled BG (Stanewsky et al., 1997a). One of the BG linescarries the transgene on chromosome 2 (BG6a) in homozy-gous condition, and the other has a homozygous-lethal BGinsert on chromosome 3 (BG/TM6B, Tb; TM6 is a marked,multiply inverted third-chromosome balancer). For obser-vations of GFP expression under the control of per-gal4 ortim-gal4 in cycle01 (cyc01) or ClockJrk (ClkJrk) mutant ge-netic backgrounds (cf. Allada et al., 1998; Rutila et al.,1998), genetically stable strains were generated in whicha chromosome 2, carrying UAS-gfp and tim-gal4 (86) orper-gal4 (2) transgenes, was balanced by CyO; and chro-mosome 3 carried either the cyc01 or ClkJrk mutation inhomozygous condition. A disconnected mutant (disco1)was used in conjunction with reporter-mediated expres-sion of tim in larvae.

Histology and visualization of clock geneproducts and marker proteins

In situ expression patterns of proteins encoded by clockgenes, marker genes, or both were assessed as detailed inTable 1.

Drawing and quantification of tissue images

To generate camera lucida-like drawings from confocalsections, images from a series of sequential 2.16-mm sec-tions were opened by applying Adobe Photoshop software(Macintosh version 4.0.1; Adobe Systems Inc., MountainView, CA). For each such section, cell bodies and processesof each neuronal cluster were traced by using differentcolors for the separate clusters. Then the separate layers’worth of drawings were merged from the first section tothe last one.

For computations involving stained areas in the retinasof adults expressing TAU under the control of per-gal4,images of immunostained head sections were first openedvia Adobe Photoshop as grayscale images. To determinethe digitized grayscale values that correlate with TAU-expressing portions of the retina, areas containing stainedphotoreceptors were arbitrarily selected by using theMagic Wand tool incorporated in this software. This toolallowed selection of clicked pixels and of the surroundingarea that exhibited an overall gray level similar to a givenlocation selected within the section. The result was ahistogram showing how many pixels exhibited each gray-scale value for the patchy, columnar-appearing areas ofstained photoreceptors selected for a given section. Thisoperation was repeated for three to four sections, for eachof three to four specimens per transgenic line. From thesehistograms, it was determined that 95% of the pixels in astained area were darker than an average grayscale valueof 119. For each section from the set just indicated, thewhole retinal area on either the left or right side of theadult head was then selected, and a histogram of gray-scale levels was determined as above. Then the proportionof pixels darker than 119 within this whole retinal areawas computed and averaged for all the sections processedfor a given per-gal4 line. This proportion was considered tobe the fraction the retina that expressed UAS-tau underthe control of per-gal4. However, in a negative controlinvolving flies that carried only UAS-tau, the proportionsof stained area darker than 119 were ca. 5% of the wholeretinal regions analyzed. Therefore, this value was sub-tracted from the average value obtained for flies carryingboth driver and marker transgenes. As a positive control,

sections from flies carrying UAS-tau and tim-gal4 (line 62)were analyzed in the manner described above and judgedto express TAU in 97% of the eye. As tim-gal4 is likely toexpress GAL4 in the entire retina, the proportions ofstained areas in eyes from per-gal4 flies were normalizedby dividing by 97%.

To calculate diameters of neuronal cell bodies near theantennal lobes, recognized in adult brains from animalscarrying per-gal4 or tim-gal4, confocal serial sections ofbrains expressing GFP under the control of either one ofthese GAL4 drivers were examined. Three to five such cellbodies, well separated from other cells, were arbitrarilyselected from each brain sample. The diameter of a givencell was measured in an optical section that showed thelargest profile of the cell. Because most cell bodies haveovoid shapes, the average of longest and shortest diame-ters for each cell was regarded as the diameter of the cell.The diameters were measured using CoMOS program(BioRad, Richmond, CA). Diameters of the cells measuredfor each sample were averaged to give a mean diameter forthat brain. Finally, the per-fly mean diameter (a mean ofa mean), 6 S.E.M., was calculated for each per-gal4 andtim-gal4 transgenic type. Four brain samples from fliescarrying both a per-gal4 (line 3) transgene and UAS-gfp inhomozygous condition were studied. Eight samples weresimilarly processed for flies carrying UAS-gfp and tim-gal4 (the latter from five separate lines) in heterozygouscondition.

Figure preparation

After initial acquisition of confocal images via CoMOS,the image files were opened by using a program calledConfocal Assistance (version 4.02). By operating the lattersoftware, confocal projections (superimposition of serialoptical sections derived from a single wholemounted brainsample) were achieved. Fluorescent TAU and TIM signals,resulting from double-labeling by antibodies to these pro-teins, were merged into colored images by using ConfocalAssistance. After these operations, images were convertedto TIF format and imported to Adobe Photoshop. In somecases, images of single optical sections were converted toTIF format without any further operations. For imagesfrom double-labeled samples, minimal adjustments of con-trast, brightness, and color balance were made via AdobePhotoshop.

GFP images of whole adults and their body parts werephotographed by using 35-mm color slide film on a ZeissAxiophot microscope equipped with fluorescent lightsource and a GFP filter. Immunostained sections werealso photographed, either as colored slides or negatives, ona Zeiss Axiophot or Axioskop. Slides and negatives werethen scanned and imported to Adobe Photoshop as TIFfiles. Color balance, brightness, and contrast for each im-age were adjusted by using Adobe Photoshop, so that theimage on screen matched the original slide or the printdeveloped from the negative. For some figures, scannedcolor images were converted to grayscale ones.

RESULTS

Clock gene-controlled GFP expression inwhole flies, larvae, and their internal organs

Whole-body expression patterns mediated by 59-flanking regions of the period and timeless clock genes


fused to GAL4-encoding sequences were assessed by ap-plication of the UAS-gfp reporter. Higher resolution anal-yses of neural structures expressing these clock genes,along with UAS-gfp or another marker, are described inlater sections.

We first examined GFP expression in animals carryingonly a UAS-gfp fusion gene. In such control adults, fluo-rescence was found in the retina; weaker signals wereobserved in the rectum, sperm pump, seminal vesicle, andovary. These fluorescent signals seem to be autofluores-cent in nature, because of their yellowish color. Signalsdependent on the per-gal4 or tim-gal4 drivers were easilydistinguished from this background, except in the adultretina, where the autofluorescence was intense. Therefore,clock gene expression patterns in the eye were assessedonly by anti-bGAL or anti-TAU immunohistochemistry onsections. In larvae, no GFP signals were detected in thedissecting microscope for control specimens carrying onlyUAS-gfp. However, in such control specimens, fluores-cence was observed by confocal microscopy in the ringgland, mouthparts, and anterior gut. Central nervous sys-tems (CNSs) from larvae, pupae, and adults carrying oneor two copies of UAS-gfp exhibited weak confocal signalsin cortical cells and at the brain surface in specimens fromthese three stages of the life cycle. Such cells were sus-pected to be glial in nature, judging by their locations andabsence of processes. Adult control brains also showedweak GFP signals in one to two cells within a ventralregion located between the optic lobe and the centralbrain. In developing animals carrying two copies of UAS-gfp, two pairs of cells in an anterior brain region and onepair in a posterior region were detected. In the larval CNS,signals were also detected in the ventral ganglion in cellsadjacent to the midline. All of these GAL4-independentGFP signals were weak and easily distinguished fromGAL4-driven expression of GFP.

For the experimental transgenics carrying such a driveralong with UAS-gfp, whole-animal and internal-organspecimens were scrutinized from three per-gal4 lines andfrom four to eight tim-gal4 lines (Table 2). The per-gal4lines chosen for these observations were those with simi-lar expression patterns amongst each other, as deter-mined in sectioned material using other reporters (seebelow). Many tissues throughout the body of adults werelabeled by GFP in flies carrying per-gal4 and UAS-gfp(Fig. 1; Table 2). The results are congruent with but moredetailed than those reported by Plautz et al. (1997a). Thespatial distribution of timeless gene products, in theirnative forms or as reported by way of markers, is under-analyzed compared with per expression patterns. Thus,we made extensive documentations of GFP-reported timexpression (Table 2). In tim-gal4 adults, the overallmarker signals were found to be similar to those driven byper regulatory sequences. However, markings of sensorycells of the head capsule and the body were not as obviousas in the case of per-gal4, because the tim-gal4-drivensignals were obscured by intense fluorescence in the fatbody underneath the cuticle (Fig. 1A).

Several internal organs in pupae and adults are knownto express the period gene. PER, a PER-bGAL fusionprotein, or bGAL driven by a 69-base-pair 59 regulatorysequence have been found in the prothoracic gland, gut,fat body, Malpighian tubules, ovary, male reproductivesystem, and the corpus cardiacum (Siwicki et al., 1988;Liu et al., 1988; Saez and Young, 1988; Frisch et al., 1994;

Emery et al., 1997; Giebultowicz and Hege, 1997; Hege etal., 1997; Plautz et al., 1997a; Hao et al., 1999). PER andTIM within internal tissues of adults are known to becolocalized only in the Malpighian tubules (Giebultowiczand Hege, 1997). Therefore, internal GFP signals drivenby per- or tim-gal4 in adults were compared. Similar over-all patterns of fluorescent marking were observed (Table2). Expression correspondence for the two clock genes wasnotable within the male reproductive system (Fig. 1D, F).However, certain differences between GFP expressiondriven by per or tim sequences were detected, especially inthe Malpighian tubules and follicle cells of the ovary. Allthe tim-gal4 lines expressed the marker, whereas none oronly one of the three per-gal4 lines drove GFP in thesetissues (Fig. 1C,E,G; Table 2).

Larval expression driven by per-gal4 or tim-gal4 wasalso compared with respect to GFP marking of non-neuraltissues. The reported spatial patterns were found not tooverlap. This was concluded by comparing the tissues thathad marker signals in common among two or more lines ofper-gal4 with those having signals in common among twoor more tim-gal4 strains (Table 2). Such labelings arelikely to reflect the normal tissue expression patterns forthese genes, as opposed to reporter signals that can resultfrom chromosomal position effects (see Discussion). There-fore, period and timeless appear not to be coexpressed innon-neural tissues at this developmental stage, whereasPER and TIM are colocalized in the larval CNS in severalneuronal clusters (Kaneko et al., 1997).

per-controlled expression in the adult brainand ventral cord revealed by application of

three reporters

This section focuses on higher-resolution examination ofperiod-gene expression patterns within the CNS. Severalof these assessments used bGAL as the marker, in speci-mens carrying per-gal4 and UAS-lacZ. Controls for theseobservations involved peroxidase-stained sections of sin-gly transgenic UAS-lacZ specimens. Background stainingwas observed in the gut, fat body, and the salivary glands.These signals may originate from the following sources:native fly peroxidase, whose enzymatic activity could me-diate the color reaction used to stain bGAL-positive cells(cf. Stanewsky et al., 1997a); leaky expression of UAS-lacZ; E. coli ingested by flies; anti-E. coli bGAL cross-reacting with the native fly enzyme (this last possibility isunlikely).

per-controlled bGAL expression was compared to thatreported by UAS-tau, visualized by anti-TAU histochem-istry. Control applications of this antibody to sections ofadult tissues in which only UAS-tau (line 24/1) waspresent led to staining in the gut, salivary glands, fatbody, the distalmost part of the retina, and a few cellswithin the abdominal ganglion. Confocal observations onwhole-mounted adult brains from the same singly trans-genic line led to anti-TAU-mediated staining of one cellwithin the lateral region of adult brain. In the larval CNSfrom this control strain, no immunoreactivity was de-tected by confocal microscopy. Background staining inperoxidase-stained adult sections from the other controltype, UAS-tau (line 7), was found in the salivary glandsand fat body. In wholemounted larval and adult brainsfrom this line, no background TAU immunoreactivity wasdetected by confocal microscopy.


For the experimental transgenics carrying per-gal4along with UAS-lacZ or UAS-tau, immunohistochemicalanalysis of horizontal sections led to results that variedamong the different per-gal4 lines. Table 3 summarizesthe marker patterns observed in six per-gal4 strains. All of

these lines exhibited reporter-gene expressions in cellsother than those known to be stained by anti-PER re-agents applied to wild-type specimens. These and otherfeatures of the bGAL- and TAU-reported results are de-scribed in the following paragraphs.

TABLE 2. GFP in Whole Animals and Non-neural Tissues Driven by per-gal4 or tim-gal4

Adult body partsand internalorgans1,2,3

Driver linesNo. specimens stained/no. observed

per-gal4 tim-gal4

1b 2 3 2712 6212 8212 8612 9 16 67 83

Body PartsAntennae 5/1010 14/14 14/14 6/6 6/6 6/6 6/6 10/10 10/10 10/10 10/10Proboscis 10/10 14/14 14/14 6/6 6/6 6/6 6/6 10/10 10/10 10/10 10/10Head capsule4 10/10 14/14 14/14 6/6 6/6 6/6 6/6 10/10 10/10 10/10 10/10Legs 10/10 14/14 14/14 6/6 6/6 6/6 6/6 10/10 10/10 10/10 10/10Wings 10/10 14/14 14/14 6/6 ND 2/2 2/2 10/10 10/10 10/10 10/10Thorax5 10/10 14/14 14/14 6/6 6/6 6/6 6/6 10/10 10/10 10/10 10/10Abdomen6 10/10 14/14 14/14 6/6 6/6 6/6 6/6 10/10 10/10 10/10 10/10

Male reproductiveorgansTestis 1 SV 1 AG7 5/5 6/6 5/5 3/3 3/3 3/3 3/3 5/5 5/5 5/5 4/4Sperm pump 7/7 3/3 0/3 6/6 6/6 6/6 6/6 5/5 5/5 5/5 4/4

Female reproductiveorgansSeminal receptacle 5/5 8/8 7/7 3/3 3/3 3/3 3/3 5/5 5/5 5/5 5/5Uterus 5/5 8/8 7/7 3/3 3/3 3/3 3/3 5/5 5/5 5/5 5/5Spermathecae 8/8 5/6 0/3 4/4 3/3 3/3 3/3 5/5 5/5 5/5 5/5AG ND ND ND 3/3 3/3 3/3 1/1 ND ND ND NDFollicle cells 0/5 0/8 0/6 3/3 3/3 3/3 3/3 5/5 5/5 5/5 5/5

Other internaltissuesMalpighian tubule 10/10 0/13 0/12 6/6 6/6 6/6 6/6 10/10 10/10 10/10 10/10Gut (midgut)8 6/6 0/611 6/6 6/6 6/6 6/6 6/6 10/10 10/10 10/10 10/10Fat body9 16/16 20/20 20/20 12/12 12/12 12/12 12/12 10/10 10/10 10/10 10/10

Larvalorgans13

per-gal4 tim-gal4

1b12 212 312 2712 6212 8212,25 8612 9 16 67 83

Tissue type14

Gut 6/616 6/621 6/621 6/623 7/723 7/726 6/623 ND ND ND NDEpidermis 6/617 0/6 0/6 6/624 0/7 0/8 0/6 ND ND ND NDSalivary glands 6/618 6/622 6/622 0/6 0/7 0/8 0/6 ND ND ND NDMalpighian

tubules3/619 0/6 0/6 6/619 1/719 0/8 0/6 ND ND ND ND

Mouth 6/620 0/6 0/6 6/620 7/720 0/8 6/620 ND ND ND NDRing gland 0/6 0/6 0/6 6/6 7/7 8/8 6/6 ND ND ND NDFat body 0/6 0/6 0/6 6/6 0/7 0/8 6/6 ND ND ND NDTrachea 6/6 0/6 0/6 0/6 0/7 4/6 0/6 ND ND ND NDSpiracles15 6/6 0/6 0/6 0/6 0/7 0/8 0/6 ND ND ND ND

1 Abbreviations: SV, seminal vesicle; AG, accessory glands; ND, not determined.2 Progeny of crosses between a GAL4 driver and UAS-gfp were observed unless otherwise stated.3 For each GAL4-driver line and for each sex, three to 10 animals were observed depending on the tissue examined. Exceptions to this were wings and female accessory glandsfor which only one or two animals were observed in some GAL4-driver lines.4 Cells near sensilla on the head capsule.5 Cells near sensilla on the thorax.6 Cells near sensilla on the abdomen.7 Signals in the testis, SV, and AG were not distinguished in some of the lines observed; therefore, animals with signals in at least one of these structures were regarded as stained;however, GFP signals in all the three male-specific organs were recognized in per-gal4 (lines 2 and 3), and tim-gal4 (lines 9, 16, 27, 67, 82, 83, and 86). The other GAL4-driver lineswere not examined in detail for these male-specific organs.8 Midgut in the abdomen.9 Fat body in the abdomen.10 Five animals with antennal signals were males, and five others without the signals were females; subsequent observations of males and females each carrying two copies foreach of per-gal4 and UAS-gfp revealed antennal expression of GFP in both sexes.11 Although there were no GFP signals found in the midgut of this particular line, signals in the rectum was observed as in per-gal4 (line 1b), and all the tim-gal4 lines; per-gal4(line 3) was not checked for rectal expression of GFP.12 Instead of progeny of crosses between a GAL4 driver and UAS-gfp, animals from established strains carrying a CyO balancer chromosome and another chromosome 2 carryingboth GAL4 driver and UAS-gfp were observed.13 For each line, three to five males and two to three females were observed.14 In addition to structures listed below, a thin membrane-like tissue located between the left and right eye-antennal discs were stained in all the three per-gal4 lines.15 Anterior and posterior spiracles.16 Spot-like signals in the entire midgut and a stripe of signal in the proventriculus were observed.17 Signals were associated with the denticle belts.18 The entire glands were stained.19 Spot-like signals were observed.20 The posterior region of the mouth part was stained.21 Signals were observed in the proventriculus and in a narrow restricted region of the midgut where the gut is most constricted.22 Proximal regions of the salivary glands exhibited stronger staining than did distal regions.23 The anteriormost midgut and a narrow restricted region of the midgut located slightly anterior to the region where GFP signals were observed in per-gal4 (lines 2 and 3).24 The entire epidermis was stained.25 A pair of cells anterior to the mouthpart was also stained.26 The anterior most midgut and hindgut were stained.


Three of these six lines gave especially variable results,while the other three per-gal4 lines (1b, 2, and 3) werefound to give similar patterns (Table 3). We interpret theunexpected signals that have little or nothing in commonamong the interstrain expression patterns to be the resultof position effects of the varying per-gal4 insertion sites

(see Discussion). Stated another way, three of the sixper-gal4 insert sites in question lead to GAL4 in normalper-expressing cells because of the per-regulatory se-quences present. Therefore, the patterns for these threelines are the main ones described below.

Marker-gene expression in PER-expressing cells.

Laterally located neuronal clusters (LNs) and dorsallylocated ones (DNs) in adults carrying per-gal4 were la-beled by both bGAL and TAU immunohistochemistry (Ta-ble 3). Expression of per in these LN and DN cells ischaracteristic of the wild-type patterns. Among the differ-ent clusters of lateral and dorsal neurons, the more dor-sally located LNs (LNds), ones with small somata (smallLNvs), and the DN2s were less reliably labeled than werethe other neuronal clusters (Table 3).

Native PER is also found throughout the retina in wild-type. However, retinal signals in the transgenics wereweak, and only ca. 30% of the photoreceptor cells werestained in flies carrying one copy each of per-gal4 andUAS-tau (Fig. 2I–K). per is normally expressed in manyCNS glia. But glial expression was nearly undetectable inthe transgenics except for weak signals in cells located atthe surface of the brain in one of the three per-gal4 lines.A putative extraretinal photoreceptor was discovered byHofbauer and Buchner (1989). It is located in a distalregion of the optic lobe complex and has been analyzed interms of anatomy as well as neurochemical cell differen-tiation by Yasuyama and Meinertzhagen (1999). Thisstructure was labeled in the three per-gal4 lines (Fig. 2A),as it is by anti-PER in wild-type (Konopka et al., 1994).

Novel neurons and their projections. Expression ofGAL4 driven by the per promoter region was detected inCNS cells beyond those known to contain native productsof the period gene. These observations involved severalclusters of neurons marked in three of the per-gal4 linesthat exhibit similar expression patterns among eachother, as well as in the line that gives ectopic trachealexpression. The neurons marked in these transgenicstrains, but not stained by anti-PER in wild-type, were asfollows: cells located dorsolateral to the antennal lobe inthe anterior cortex; a cluster of neurons ventrolateral tothe antennal lobe in the anterior cortex; cells near thetritocerebrum and the subesophageal ganglion; large andsmall cell bodies in the posterior cortex; a pair of cells atthe esophageal foramen; and cells in the pars intercereb-ralis (Table 3; Fig. 2A,C,E–H; cf. Strausfeld, 1976). All

Fig. 1. GFP expression in the whole fly body and in various adulttissues, driven by tim-gal4 or per-gal4. All flies carried one copy of theUAS-gfp marker transgene. A: Whole body of an adult male carryingone copy of tim-gal4 (line 62); fluorescence, emanating from the entirebody and its appendages, mainly comes from the fat body and sensorycells. B: Cells at the wing margin of a fly with the same genotype asin A. C: Malpighian tubules from a fly of the same genotype as in Aand B D: Male reproductive system and rectum of a fly carrying onecopy of tim-gal4 fly (line 27). E: Female reproductive system andrectum; same tim-gal4 strain as in D. F: Male reproductive systemand rectum of a per-gal4 fly (line 2). G: Female reproductive systemand rectum of per-gal4 (line 2); per-controlled signals in the uterusand the spermathecae were weak in the microscope and are difficult todiscern in this photograph. AG, accessory gland; ED, ejaculatory duct;F, follicle cells; O: ovary; R, rectum; SP, sperm pump; SPT, sper-mathecae; SR, seminal receptacle; SV, seminal vesicle; T, testis; U,uterus; VP, vaginal plate. For other abbreviations, see list. Scale bar5 100 mm in A,C,D (also applies to E–G); 200 mm in B.


neuronal types in the locations just listed were found inthree or four strains, except for cell bodies in the posteriorbrain cortex. Here, the positions of stained cell bodieswithin the posterior cortex were more widely variableamong the per transgenic strains (Table 3). Therefore,most of these neuronal signals, except those in the poste-rior cortex, seem to be due to per-promoter activity.

Fibers from the neurons that have not been found tocontain native PER, but were observed in multiple per-

gal4 lines, were traced in sectioned material by TAUmarking and the examination of TAU- or GFP-labeledwholemounts. Among these fibers, the most conspicuousones are in the ellipsoid body (EB) of the central complex(Fig. 2A,E,G,I,J; cf. Hanesch et al., 1989) and the relayinterneurons of the antennal lobes coursing within themiddle antennocerebral tract (mACT; Fig. 2C,F,I–K; cf.Stocker et al., 1990). The former originates from cell bod-ies located dorsolateral to the antennal lobes (Fig. 2A,E, J)

TABLE 3. Cells, Neurites, and Tissues Expressing GAL4 in per-gal4 Transgenic Lines Revealed by TAU or bGAL markers1,2,3

per-gal4 linesNo. specimens stained/no. observed)

1b 2 3 0 1a19 523

per-expressing neurons (cell bodies)Large LNv 7/7 8/8 5/5 5/9 ND20 ND17

Small LNv 0/7 8/8 4/5 6/9 ND20 ND17

LNd 0/7 8/8 2/4 4/9 ND20 ND17

DN1 7/7 8/8 5/5 9/9 ND20 ND17

DN2 3/8 4/8 1/4 0/9 ND20 ND17

DN3 3/8 8/8 5/5 8/9 ND20 ND17

per-expressing neuronal fibersPOT 7/8 8/8 5/5 3/8 ND20 ND17

Medulla4 8/8 8/8 5/5 4/8 ND20 ND17

DN1/small LNv5 7/8 8/8 5/5 ND16 ND20 ND17

Dorsal commissure 8/8 8/8 5/5 9/9 ND20 ND17

Other per-expressing structuresOcelli 2/512 6/612 1/512 3/3 4/4 2/4Retina (photoreceptors) 8/812 8/812 5/512 9/912 4/412 6/6H-B photoreceptor 1/7 8/8 4/5 ND17 ND20 ND17

Glia 0/8 0/8 5/515 9/918 ND20 6/622

Cardiac epithelial cells 8/812 7/812 5/512 2/812 4/412 0/6Ovary6 ND13 ND13 ND13 4/4 ND13 5/5

Reporter-labeled brain neurons (cellbodies) not PER-expressing in WTPars intercerebralis 6/6 8/8 0/4 8/8 ND20 ND17

Small cells in posterior cortex 8/8 8/8 3/5 9/9 ND20 ND17

Large cells in posterior cortex 0/7 8/8 0/5 0/9 ND20 ND17

Cells dorsal to AL (EB cells) 8/8 8/8 5/5 9/9 ND20 ND17

Cells ventral to AL (mACT) 8/8 7/7 5/5 9/9 ND20 ND17

Cells near lobula 0/8 8/8 0/4 4/8 ND20 ND17

Esophageal foramen7 6/8 5/6 3/4 4/9 ND20 ND17

TRI/SEG8 5/7 8/8 3/3 7/9 ND20 ND17

Lamina neurons 8/8 0/8 0/5 ND17 ND20 0/6Reporter-labeled brain neurons (fibers)

not PER-expressing in WTCentral complex (EB and FB) 8/8 8/8 5/5 9/9 ND20 5/5Antennal lobe 8/8 8/8 5/5 9/9 ND20 6/6Lateral horn9 8/8 8/8 5/5 9/9 ND20 5/5

Reporter-labeled structures in the bodynot PER-expressing in WTThoracic muscles 0/8 0/8 0/5 0/9 0/4 6/6Trachea10 0/8 0/8 0/5 9/9 0/4 0/6Thoracic ganglia (cell bodies) 4/414 4/414 1/114 8/814 3/321 ND17

Thoracic ganglia (fibers) 4/4 6/6 2/2 8/8 3/322 5/5CC (fibers)11 4/4 6/6 4/4 6/6 3/322 0/5

1 Numbers of samples observed for each per-gal4 line were as follows: line 1b, four samples in combination with UAS-tau (line 24/1) and four with UAS-lacZ; line 2, five withUAS-tau (24/1) and three with UAS-lacZ; line 3, one with UAS-tau (24/1) and four with UAS-lacZ; line 0, nine with UAS-lacZ; line 1a, four with UAS-lacZ; line 5, six with UAS-lacZ.Certain tissues and cells were missed because of the loss of some sections in certain specimens. Thus, the numbers stained are given as numerators. Numbers of samples observedare given as denominators for each cell type, fiber, and tissue.2 Abbreviations: CC, corpus cardiacum; EB, ellipsoid body; FB, fan-shaped body; H-B photoreceptor, Hofbauer-Buchner eyelet; mACT, middle antennocerebral tract; POT, posterioroptic tract; WT, wild-type; ND, not determined. For other abbreviations, see list.3 The bottom three sections tabulate regions of lacZ or tau expression that originate from cells in which PER has not been previously detected.4 Varicose fibers in the distalmost layer of the medulla.5 Processes of small LNvs and DN1s that connect the region containing LN cell bodies with the superior protocerebrum.6 Follicle cells in the ovary.7 A pair of relatively large cell bodies in either side of the esophagus in the frontal cortex.8 Cortex near the tritocerebrum and subesophageal ganglion.9 Terminal arborization of the antennal lobe relay interneurons of the mACT in the lateral horn.10 Tracheal staining in the head as well as in the fly’s body.11 Fibers in the region of CC.12 Not all the cells in the tissue were stained.13 Not determined, because only males were tested.14 Cells in the region of prothoracic ganglion, mesothoracic ganglion, and metathoracic ganglion.15 Glial cells in the first optic chiasm and surface-associated glia near the periphery of the brain.16 Tracheal expression masked the signals.17 Glial expression masked the signals.18 Surface glia were stained.19 Numerous neurons in the central nervous system (CNS) were stained.20 Staining of numerous neurons masked the observability of cell bodies and neurites in this line.21 Many cells in the thoracic ganglia were stained.22 The entirety of these tissues were stained.23 Numerous glial cells in the cortex were stained.


Figure 2


and the latter from cells ventrolateral to the antennallobes (Fig. 2A,E).

Details about these fibers and others that originate fromnon-PER-expressing neurons are as follows: The neuronsdorsolateral to the antennal lobes can be divided into twogroups, a larger one located dorsomedial to a smaller one.Neurons in both clusters project to the central complex(Fig. 2A,E,G,I,J) with intense arborization in the EB andthe lateral triangle. This pattern of arborization is similarto that of anti-gamma aminobutyric acid (GABA)-immunoreactive ring neurons described by Hanesch et al.(1989). For the current analysis, the neuronal cluster lo-cated ventrolateral to the antennal lobe was subdividedinto two clusters, a larger one located dorsal to a smallerone. Neurons in the large cluster, located in a relativelydorsal brain region (Fig. 2A,E), correspond to the relayinterneurons of the antennal lobe that take the pathway ofthe mACT. Neurons in the smaller, more ventral clusterproject medially to the anterior region of the tritocere-brum (below the esophagus in Fig. 2A,E), apparentlywithin the gustatory center (cf. Stocker, 1994). Thus,these particular per cells may correspond to interneuronswithin this sensory center (cf. Nayak and Singh, 1985).Some of the labeled cell bodies near the tritocerebrum andthe subesophageal ganglion seem to be descending neu-rons that project to the thoracic nervous system (cf.Strausfeld et al., 1984). Certain such cells project dorsallyto the superior protocerebrum, and others send axonsthrough the pharyngeal nerve or the accessory pharyngealnerve (not shown; cf. Rajashekhar and Singh, 1994). Apair of cells in the esophageal foramen (Fig. 2E) projectsalong the esophagus posteriorly. In the posterior region ofthe brain, this fiber appears to turn laterally, sending afiber parallel to the posterior optic tract (POT, cf. Straus-feld, 1976) and entering the optic lobe (Fig. 2C,F). Fibersof neurons in the pars intercerebralis (Fig. 2A,H; cf.

Strausfeld, 1976) follow the median bundle ventrally butwere not traced beyond the esophagus, because too manyfibers were labeled just below the esophagus.

Stained fibers in the antennal lobe (Fig. 2A,E,K) seem tooriginate from the mACT interneurons and not from thesensory neurons of the antennae, because no immunore-activity was found in the antennal nerve in the threeper-gal4 lines whose expression patterns were consistentamong each other (data not shown). Therefore, the anten-nal signals reported by GFP in these three lines (seeabove) probably do not come from neuronal cells. The lackof signals originating from neurons in the antennae wasunexpected, given that circadian rhythms have been de-tected in this organ’s electrophysiological responses toodorants (Krishnan et al., 1999).

In the thoracic and abdominal ganglia, the period genehas been reported to be expressed only in glial cells (Eweret al., 1992). Here, glial expression of markers was notdetected in the three mutually consistent per-gal4 linesand in two others. Instead neuronal cell bodies and fiberswere stained in these five lines (Table 3).

Traced fibers from putative PER-expressingneurons in the adult brain

Because all classes of PER-immunoreactive neuronswere revealed by per-gal4 (see below), this construct wasused to map the projection patterns of these neurons. Forthis purpose, stable strains carrying one copy of per-gal4(line 2) and two copies of UAS-tau were subjected towholemount anti-TAU immunofluorescence staining andviewed by confocal microscopy (Fig. 2A–D,G,H). Immuno-stained horizontal sections of flies carrying per-gal4 andUAS-tau or UAS-lacZ were compared with the confocaldata (Table 3; Fig. 2I–K). Adult brains from flies carryingper-gal4 (line 2 or 3) along with UAS-gfp were also ob-

Fig. 2. GAL4 expression in adult brains of per-gal4 transgenics,revealed by TAU or GFP markers. A,C,E–H: Thirty to seventy confo-cal sections at 2.16-mm depth intervals are superimposed. White orblack arrows in A, C, E–K, cell bodies of known PER-expressingneuronal clusters; arrowheads, fibers projecting from these cells;green arrows and arrowheads, cell bodies and their fibers, respec-tively, of neurons not previously detected with anti-PER. A: Frontalview of a brain hemisphere from an adult carrying one copy of per-gal4(line 2) and two copies of UAS-tau (line 24/1); 1, DN1 cells; d, LNds; lv,large LNvs; sv, small LNvs; H-B, fibers of Hofbauer-Buchner eyelet;EB, neurons projecting to the ellipsoid body and EB neuropil itself; AI,antennal lobe relay interneurons; TRI, cell bodies located near thetritocerebrum; PI, pars intercerebralis; AL, antennal lobe; Eso, esoph-agus. B: Diagram of TAU expression driven by per-gal4 (as shown inA), depicting positions of known PER-expressing neuronal somataand their projections; color coding for separate cell body clusters andtheir neurites: dark blue, DN1 cells; red, DN3s; green, LNds; purple,large LNvs; light blue, small LNvs pink, H-B eyelet. C: TAU expres-sion driven by per-gal4 in a posterior region of same brain hemisphereshown in A; 1, putative DN1 cells; 3, DN3s; POT, posterior optic tract;SEG, cell bodies near the subesophageal ganglion; leftmost greenarrow, neuron near the DN3 cluster that projects ventrally to a largecell body in the posterior cortex (green arrow in the middle); upper-most green arrow, neuron projecting ventrally to the esophagus (Eso)from region of DN1 neurites; AI, fibers of antennal relay interneurons;EF, cells at the esophageal foramen that lack PER-immunoreactivity;unlabeled green arrowheads, putative descending fibers originatingfrom cell bodies near the SEG. D: Diagram of TAU expression drivenby per-gal4, depicting positions of known PER-expressing neuronalsomata and their projections (as shown in C); color coding as in B,

with the addition of yellow for neurite of the ventrally projecting cellwhose marker signal terminates near the esophagus. E: Anterior sideof a brain hemisphere from a fly carrying per-gal4 (line 3) and UAS-gfp (two copies each); structure labeling as in A and C. F: GFP drivenby per-gal4 in a posterior view of the same brain shown in E; 1 witharrowhead, processes of putative DN1s; L Ho, lateral horn; otherstructure labeling as in A and C. G: Horizontal view of the dorsalregion of a brain carrying per-gal4 (line 2, one copy) and UAS-tau (line24/1, two copies); structure labeling as in A; asterisk, debris. H: TAUexpression driven by per-gal4 (same genotype as G) in the superiorprotocerebrum; structure labeling as in A, including depiction of LNd

neurites that are similar to the projection pattern of the dorsal giantinterneuron; left of thin green line, confocal projection from anteriorside of brain, juxtaposed with projection from posterior side on theright. I: Horizontal section of a brain carrying one copy each ofper-gal4 (line 2) and UAS-tau (line 24/1); this TAU-immunostainedsection is at the level of the ellipsoid body (EB) and fan-shaped body(FB) in the brain’s central complex; d, LNd cell body; sv and 1, fiberbundles from small LNvs and from DN1s, respectively; AI, antennallobe relay interneurons; LTR, lateral triangle; Ol, optic lobe complex;R, retina. J: TAU immunostaining of a section of the same fly as in Iat the level of the neuronal somata projecting to the EB; structurelabeling as in I. K: TAU immunostaining of a section from the same flyas in I at the level of the large LNv cells and the esophagus (Eso); lvwith arrow, large LNv cell body; lv, sv, and 1 with arrowheads, fibersprojecting from large LNvs, small LNvs, and DN1s, respectively; un-labeled arrowheads, distal- and proximalmost layers of the medullaoptic lobe. For other abbreviations, see list. Scale bars 5 50 mm in A,H(applies to A–H); 100 mm in I (applies to I–K).


served in this manner (Fig. 2E,F). These per-gal4 lines areamong those with similar expression patterns (Table 3).

Ventral LNs with large somata. As has been shown byanti-PDH immunohistochemistry (Helfrich-Forster andHomberg, 1993), fibers from the large per-expressing LNvcells project medially to the POT and into the medulla.The per-gal4 drivers mediated prominent POT signals,but the centrifugal projection was only faintly labeled byanti-TAU (Fig. 2A–D,J–K). However, stained fibers in thedistalmost medulla layer were found in four lines (Table 3)and in the serpentine layer in two lines (1b and 2) byobservation of immunostained sections (Table 3; Fig. 2K).In addition, medulla fibers in a layer distal to the serpen-tine layer and in a proximal region close to the lobula opticlobe were stained in sections of two lines (Fig. 2K).

Ventral LNs with small somata. Small LNv cells sendfibers dorsally to a brain region located dorsal and ante-rior to the calyces of the mushroom body (Helfrich-Forsterand Homberg, 1993). A small LNv-like bundle of fibersthat runs from the location of these cell bodies to thesuperior protocerebrum was also labeled in the doubletransgenics. However, it is unlikely that all of these pro-cesses originate from the small LNvs, because PDH-immunoreactive fibers in this region are thinner than theappearance of this bundle in the per-gal4/UAS-tau flies(Fig. 2C,D,F,I,J). Among the fibers in this bundle, oneslocated in relatively medial regions seem to form terminalendings at the superior protocerebrum, indicating thatthey originate from the small LNvs (Fig. 2D). More later-ally located fibers appear to originate from a dorsallylocated cluster of per cells called DN1 (see below). At thelevel of the esophagus, this bundle, consisting of smallLNv and DN1 neurites, seems to join the fibers from thelarge LNvs (Fig. 2D,K). Neurites from these three sourcescourse together along the inner margins of the medullaand lobula (Fig. 2K), ultimately reaching the region inwhich LN cell bodies are located. This anti-TAU-stainedfiber tract appears to be part of Cuccatti’s bundle (Fisch-bach and Dittrich, 1989).

Dorsal LNs. Fibers from the dorsal group of lateralneurons, called LNd cells (Fig. 2A,E), were observed in thetransgenics to project ventrally for a short distance, turndorsally to make a characteristic loop near the cell bodies,then course dorsally and medially along the distal surfaceof the lateral horn (Fig. 2A–D,G,H; cf. Strausfeld, 1976).Although both small LNvs and LNds terminate in a dorsalbrain region, fibers of LNds were observed to take a routemore anterior and lateral to those of small LNvs. Afterthey turn medially in the superior protocerebrum, neu-rites from the LNd cells course medially and in a slightlyventral direction. In five of 12 TAU-labeled samples forwhich LNds were strongly labeled, bifurcation of thesefibers along their way to the contralateral side of the brainwas observed (Fig. 2B,D). This projection is very similar tothe one documented for the dorsal giant interneuron (DGI)as observed in certain GAL4 enhancer-trap lines (Ito etal., 1997b). In fact, a large cell body similar to the DGI wasintensely labeled by anti-TAU near the LNd cluster (e.g.,Fig. 2A). However, this neuron was not labeled by anti-TIM in samples double-labeled by anti-TAU (see below).Therefore, signals of the bifurcating fibers that projectmedially and cross the midline may originate mainly fromthe DGI but not from the LNds, even though LNd fibersappeared to take the same pathway as the DGI neurites.The loop near the LNd cell bodies and the bend outside the

lateral horn originate both from the LNds and the DGI,because this fiber bundle consisted of more than two fibersat least in seven of 12 TAU-immunostained brain samples(data not shown).

Dorsal neurons, group 1. From the DN1, cell bodieslocated in the dorsalmost cortex, fibers were observed toproject ventrally for a short distance to the region whereaxons from the small LNvs terminate (Fig. 2C,D,H). Manyof these neurites then extend medially and make aV-shaped commissure in the superior protocerebrum (Fig.2A–D,F–H). This commissure is located just above andanterior to the calyces of the mushroom body. Althoughsome of the DN1 fibers may cross the midline and form thecommissure, others seemed to terminate near the midline.In addition, a few fibers originating in the region of theDN1 cluster were seen to make a lateral projection ini-tially, then an anterior turn at the lateral superior proto-cerebrum, followed by a medial extension back to thedorsomedial protocerebrum. This loop of putative DN1fibers was found in four of 12 TAU-immunostained brains(Fig. 2G). Above the mushroom body calyces, where thefibers from the small LNvs terminate, processes fromDN1s seem to join the small LNvs (Fig. 2C,D) and extendventrally toward the anterior rim of the medulla (alsoknown as the accessory medulla; Helfrich-Forster, 1997).It could not be determined whether these fibers originatefrom cell bodies located in the ipsilateral or the contralat-eral hemisphere. It was also not clear where these fibersterminate, although they appeared to enter the optic lobe(Fig. 2D). Such fibers might correspond to those observedin sectioned heads distal to the serpentine layer or in themost proximal region of the medulla (Fig. 2K).

From the commissure of the DN1s, a few fibers projectventrally along the posterior surface of the brain (Fig.2C,D,F). After such fibers reach the esophagus, no neuritesignals extended beyond that alimentary structure (Fig.2C,D). This pathway is reminiscent of neurosecretory cellsin other insects, especially those in the nervi corporiscardiaci 1 and 2 (Coopenhaver and Truman, 1986; Vee-laert et al., 1998), which innervate the complex of corporacardiacum (CC) and allatum (CA). Therefore, these neu-rites may extend further along the esophageal canal andterminate in the CC, CA, or both. In fact, varicose fiberswere recognized in the region of CC in four of six per-gal4lines, by observations of immunostained sections (Table3). At first these fibers were suspected to originate fromthe two neurons of the DN2 cluster, which sit just abovethe calyces of the mushroom body (Kaneko, 1998). How-ever, in brain samples of TAU-expressing flies double-labeled with anti-TIM and anti-TAU, TIM-immuno-reactive DN2s were rarely labeled by the TAU antibody(see below). For this reason, we judged these fibers tooriginate from one of the DN1 cells. Along these course ofthese processes, a relatively large neuron located ventralto the DN1 commissure also sends fibers ventrally to theesophagus (Fig. 2C). This neuron, however, was notstained by anti-TIM in brain samples from the flies car-rying per-gal4 and UAS-tau that were double-labeled byanti-TAU (see below).

Newly identified dorsal neurons. Among dorsally lo-cated neuronal clusters expressing the period gene, DN3is a recently discovered cell group located in the lateralsuperior protocerebrum. It was first reported to be presentwithin pupal brains (Kaneko et al., 1997). The DN3 clus-ter consists of approximately 40 neurons with small so-


mata (Fig. 3C). In adults, these dorsal neurons were ini-tially recognized by their expression of a PER-bGALfusion protein called BG (see Materials and Methods) andby anti-PER immunohistochemistry (M. Kaneko and C.Helfrich-Forster, unpublished observations). Double la-beling by anti-bGAL and anti-ELAV on adult brains froma per-lacZ fusion gene transgenic confirmed that thesecells are indeed neurons, given that the elav gene is ex-pressed exclusively in all neurons (Yao et al., 1993). Innine flies that were sectioned or had their brains whole-mounted (Table 1), all detectable DN3 cells were double-labeled (data not shown).

DN3 cells in transgenics carrying per-gal4 were ob-served to send processes medially and cross the fibers ofDN1 and small LNv cells near the lateral horn (Fig.2C,D,G,H). Most DN3 neurites seemed to extend furtherin a medial direction, below the DN1 commissure, andterminate at the pars intercerebralis (Fig. 2C,D,G,H).However, it was difficult to follow single neuronal fibers atlocations where those projecting from DN3 cells meet theDN1/small-LNv fibers near the lateral horn. Thus, it ispossible that some of the DN3 neurites take the pathwayof DN1s and join the dorsal commissure or project towardthe region in which LN cell bodies are located. We ob-served one cell in the region of the DN3 cluster of per-gal4flies, which is not immunoreactive for the timeless geneproduct (see below). This neuron sends its axon in a ven-tral direction (Fig. 2C).

In summary, processes from many neurons expressingthe period gene were revealed by TAU or GFP expression,elicited by the per-gal4 fusion gene. Several of these neu-rites were found to innervate the superior protocerebrum.Such dorsal brain termini were observed not only for thesmall LNv cells, as previously revealed by anti-PDH stain-ings (Helfrich-Forster, 1996), but also for other per neu-rons whose projections have never been examined (seeDiscussion).

Double-labeling of adult brains to detectper-gal4-controlled TAU and coexpression

of timeless

GAL4-mediated expression of TAU and GFP in fliescarrying per-gal4 and UAS-gfp or UAS-tau was examinedin neuronal clusters that putatively express clock genes inwild-type brains. Colocalization of these markers with thenative product of such genes was an important concern,because the transgenes are expressed in several locationspreviously unidentified as containing per or tim gene prod-ucts in wild-type, and because transgene-driven reporter

expression was not found in certain cells that normallyexpress these clock genes (see above). Thus, we appliedboth anti-TIM and anti-TAU to brains expressing TAUunder the control of per-gal4. Most flies were killed 3hours before lights-on (ZT21 in a 12:12 LD cycle), as this isnear the time of TIM’s normal abundance peak (Hunter-Ensor et al., 1996; Stanewsky et al., 1998). Another reasonfor labeling these specimens with anti-TIM is that there isno information on colocalization of the products of per andtim in the adult brain.

per- and tim-expressing neuronal clusters. In Table4, brain cells that were double-labeled, expressed onlyTIM, or only TAU, are enumerated for each neuronalcluster. In Figure 3, examples of double-labeled cells foreach cluster are shown. Among all the tim-expressingneuronal clusters, only the LNv cells with relatively largesomata were also invariably labeled by per-driven TAU(Fig. 3A,B). In the other clusters, some of the cells labeledby TIM were not labeled by anti-TAU (Fig. 3A–F). In thesmall LNv cluster (Fig. 3A,B) and that of DN2 cells (Fig.3D), fewer than half of the TIM-expressing neuronsstained for TAU. For all the clusters except those of DN2neurons, cells labeled only by anti-TAU were detected inclose proximity to those expressing TIM (Table 4). Amongthese TIM-negative but per-gal4-expressing neurons,there was a relatively large cell body near the LNd somatathat sends its axon within the projection from TIM-expressing LNds (data not shown). This cell may corre-spond to the dorsal giant interneuron, because of its largecell body (see above and Ito et al., 1997b). In addition tothis putative DGI, one cell near the LNd cluster stained forTAU in the control strain that carries only the UAS-tautransgene. The nearby cells may be rhythm-related, eventhough they were found to express only the per gene inthis particular assay. The lack of detectable TIM immu-noreactivity in such neurons could have occurred eitherbecause this protein is not abundant enough at this par-ticular time point or because the cells simply do not ex-press tim. Although their numbers within the DN3 clusterwere not counted, at least some of the cells were double-labeled, whereas others were stained only by anti-TIM oranti-TAU (Fig. 3D,E). This is the same kind of resultfound for small LNv cells, LNds, DN1s, and DN2s.

A novel TIM-immunoreactive cell cluster. A smallgroup of cells previously unknown to express the timelessgene was found in the posterior lateral cortex of the pro-tocerebrum, in samples double-labeled by anti-TAU andanti-TIM (Fig. 4A). This three-cell cluster was subse-quently detected in sections of wild-type flies stained by

TABLE 4. Double-Labeling With Anti-TIM and Anti-TAU Applied to Adult Brains From Flies Carrying UAS-tau and per-gal41

Cell type No.2Double-labeled

(%)3TIMonly4

TAUonly5

TIM(total)6

TAU(total)7

Large LNv 17 (9) 3.8 6 0.1 (100) 0 6 0 0.6 6 0.2 3.8 6 0.1 4.4 6 0.2Small LNv 17 (9) 2.1 6 0.2 (48) 2.4 6 0.2 0.8 6 0.3 4.4 6 0.2 2.9 6 0.4LNd 16 (9) 4.5 6 0.3 (78) 1.3 6 0.3 3.8 6 0.5 5.8 6 0.1 8.3 6 0.5DN1 14 (7) 7.4 6 0.4 (57) 5.6 6 0.4 5.1 6 0.6 12.9 6 0.5 12.5 6 0.8DN2 14 (7) 0.4 6 0.2 (36) 0.8 6 0.2 0 6 0 1.1 6 0.2 0.4 6 0.2

1 Flies carrying two copies of UAS-tau (line 24/1) and one copy of per-gal4 (line 2) were used; the genetic background included the normal tim1 allele.2 Numbers of brain hemispheres observed (numbers of brains in parentheses).3 Numbers of cells double-labeled (6S.E.M.) for each tim-expressing neuronal cluster per brain hemisphere (in parentheses, the percentages of double-labeled cells among the cellsexpressing TIM within a given cluster).4 Numbers of cells (6S.E.M.) labeled only by TIM.5 Cells labeled only by TAU.6 Total numbers of cells labeled by TIM.7 Total numbers of cells labeled by TAU.


anti-TIM. The signals were robust at ZT21 (Fig. 4B), butweak at ZT9 (not shown). This suggests that TIM proteinundergoes circadian cycling or at least light-driven oscil-lations in these cells. They have never been identified asTIM expressors (Hunter-Ensor et al., 1996; Stanewsky et

al., 1998), nor were they observed previously via anti-PERstaining or bGAL reporting mediated by a per-lacZ fusiongene (Liu et al., 1988; Siwicki et al., 1988; Ewer et al.,1992; Stanewsky et al., 1997a). Whether these cells areglial or neuronal in nature is not known. However, in

Figure 3


brain samples from flies carrying per-gal4 and UAS-taudouble-labeled by anti-TIM and anti-TAU, weak TAU im-munoreactivity was detected in one or two of the threeTIM-positive cells in six of 14 hemispheres examined (Fig.4C). This line of per-gal4 (2) drives little glial expression ofmarker genes, so these cells may be novel per- as well astim-expressing neurons.

Non-PER-expressing cells. We paid special attentionto TAU-labeled cells that have not previously been iden-tified to contain per or tim gene product. For this, TIMimmunoreactivity was scrutinized in two to 13 brain hemi-spheres (depending on the cell cluster) that had beensubjected to double-labeling. As a result, the followingcells were found to express TAU only: neurons near theantennal lobe that project to the ellipsoid body of thecentral complex (Fig. 3B); the antennal lobe relay inter-neurons of the mACT; cells in the pars intercerebralis;large cells in the posterior protocerebrum; one located inthe posterior protocerebrum, whose neurite fasciculateswith the DN1s to the esophagus along the posterior sur-face of the brain; cells located ventral to the mACT neu-rons in the anterior cortex; and a cell near the DN3s thatprojects ventrally (see above). Although per-expressing,TAU-positive cells near the subesophageal ganglion and

Fig. 3. Adult brains expressing UAS-tau under per control, double-labeled by anti-TAU and anti-TIM. Each panel displays a singleconfocal section from a fly carrying one copy of per-gal4 (line 2) andtwo copies of UAS-tau (line 24/1). For a given trio of lettered panels,signals mediated by anti-TIM are on the left, those by anti-TAU in themiddle, the merged image on the right (TIM signals in green, TAU inred). A: Lateral brain region (dorsal at the top), containing threedouble-labeled large LNv cells (lv); one of two TIM-expressing smallLNv cells (sv) was also labeled by anti-TAU; TAU immunostaining isweaker than the TIM signal in this double-labeled cell (small arrow onthe right), such that the red signal is masked by the green one. B:Lateral brain region (dorsal at the top), containing two double-labeledlarge LNvs (lv) and two such small LNvs (sv); the latter cells weaklyexpressed the two antigens (small arrows on the right); in the middlepanel cells that project to the ellipsoid body (EB) were not double-labeled. C: Dorsolateral brain region (dorsal at the top), containing sixLNd cells (d), five of which were double-labeled; three additional cellswere labeled by anti-TAU. D: Dorsoposterior brain region (dorsome-dial at the top), including two DN2 cells (2), one of which (on the right)is double-labeled; among the DN3 cells (3), some are double-labeled,some only by anti-TIM, others only by anti-TAU. E: Dorsoposteriorbrain region (13.0 mm anterior to the one in D) containing two double-labeled DN1 cells (1); some of the DN3s (3) are double-labeled, andothers expressed one or the other antigen. F: Dorsoposterior brainregion (4.3 mm anterior to the one in E), containing three double-labeled DN1s (1), one labeled only by anti-TIM (small arrows), andone only by anti-TAU (small arrowheads). For abbreviations, see list.Scale bar 5 50 mm (applies to A–F).

Fig. 4. A novel cluster of TIM-containing cells in the posterolateralbrain. All specimens came from flies carrying one copy of per-gal4 (line2) and two of UAS-tau (line 24/1). A: Wholemounted adult brain,double-labeled by anti-TIM and anti-TAU; 34 confocal sections at2.16-mm depth intervals are superimposed (dorsal is at the top); onlyTIM immunoreactivities in the dorsal cortex of this brain hemisphereare shown; 1, DN1 cells; 2, DN2s; 3, DN3s; arrow, the novel three-cellcluster. B: The novel cluster in a horizontal adult head section, singly

labeled by anti-TIM; arrow, the three-cell cluster; arrowheads, glia; R,retina. C: Confocal section of posterodorsal region (lateral at the top)of a wholemounted adult brain; staining mediated by anti-TIM is onthe left, by anti-TAU in the middle; the merged image on the right(TIM signals in green, TAU in red); unlabeled arrow, a double-labeledcell within the novel cluster; 1, DN1s; 3 with arrowheads, DN3s; 3with arrows, DN3 fibers; sv, 1, fibers from small LNvs and DN1s;green arrows, non-TIM-expressing cells. Scale bars 5 50 mm.


the tritocerebrum were not checked for TIM immunoreac-tivity in these samples, only weak signals were detected inthis region by anti-TIM single-labeling of sections (datanot shown).

Patterns of reported timeless expression intim-gal4, neurite marker transgenics

Spatial expressions of tim-controlled GFP in adultbrains from flies of eight tim-gal4 lines were assessed viaconfocal observations of wholemounted samples. Three ofthese lines (62, 82, and 86) were also studied in combina-tion with UAS-tau (line 7) by anti-TAU immunohisto-chemistry. Of the three genotypes stained with this anti-body, two were observed in fluorescently stainedwholemounted samples as well as in peroxidase-stainedsections, and one line by sectioning only. Expression pat-terns revealed by all the methods were essentially identi-cal, except that GFP is relatively concentrated in thenuclei compared with TAU, which was exclusively ex-tranuclear.

tim-gal4-driven marker patterns in adult brain cells.

Expression patterns of GFP and TAU were found to besimilar to that of endogenous TIM protein, except thatGFP and TAU signals are more cytoplasmic than the TIMsignals detected by immunohistochemistry in wild-typespecimens (Hunter-Ensor et al., 1996; Stanewsky et al.,1998). Strong TAU signals were observed in thecompound-eye retina and the ocelli of all three lines whosesectioned specimens were stained by application of anti-TAU (Fig. 5E–G; ocellar signals not shown). Numerousglia throughout the brain were also marked by GFP, TAU,or both (Fig. 5A–E). These glial signals were detected atthe surface of the brain, in the cortex, between cortex andneuropil, and at the borders between adjacent neuropilstructures. Because PER- and TIM-immunoreactive glialcells are also found in these regions within wild-typebrains, glia that express these clock genes seem to belabeled by TAU or GFP in flies carrying tim-gal4 andUAS-tau or UAS-gfp.

These glial signals mediated by TAU or GFP maskedthose present in neuronal clusters and made it impossibleto trace their fibers. However, several cell body clusterssimilar to per/tim-expressing ones were detected by TAUor GFP. Among these neuronal clusters are large andsmall LNv cells, LNds, and DN1s (Table 5; Fig. 5A,D).Neuronal fibers from these neurons, similar to those la-beled under the control of per-gal4, were found in thefollowing locations: the POT originating from large LNvsomata (Table 5; Fig. 5C); processes to the superior pro-tocerebrum from the small LNvs and collaterals from theDN1s (Table 5; Fig. 5C,F); fibers projecting from LNds thatmake a loop near the cell bodies (Table 5; Fig. 5C); and thedorsal commissure of DN1 neurites (Table 5; Fig. 5D).

Novel tim-expressing cells in adult brains. In addi-tion to the cells that contain native TIM protein (Fig. 3),two neuronal clusters were recognized by GFP expressionin the tim-gal4 transgenics (Fig. 5A). One such cluster islocated dorsolateral to the antennal lobe (observed in 28 of34 brains), the other near the tritocerebrum and the sube-sophageal ganglion (35 of 38 brains). Consistent withthese observations, weak and diffuse staining was ob-served in these cells near the antennal lobes in sections ofwild-type flies stained by anti-TIM, both at ZT21 and ZT9(data not shown). Although the positions of the cell bodiesnear the antennal lobes were similar to the ones labeled in

per-gal4-containing flies (Fig. 2A,E), many of these newlynoticed tim cells seem to be different: the perikarya intim-gal4 were larger than those in per-gal4 (respectively,5.9 6 0.3 mm vs. 3.8 6 0.1 mm). Also, only weak signalswere detected in the ellipsoid body in three of the eighttim-gal4 lines. For the five lines in which no EB signalswere detected, cell bodies near the antennal lobes werefound (compare Figs. 5E and 2I, which depict sectionsfrom a similar brain level). Therefore, signals in these cellbodies do not correlate with the EB signals in tim-gal4lines. GFP or TAU signals controlled by tim-gal4 in theantennal lobe neuropil may originate from this neuronalcell cluster near this lobe but not from the olfactory recep-tor neurons in the antenna (Fig. 5A,G). This is because theweak and diffuse signals observed in the antennal nervesseem to be glial in nature, indicating that the olfactoryreceptor neurons do not express tim. This lack of tim-gal4-mediated reporter expression in the neurons of the anten-nae is consistent with the lack of such signals mediated byper-gal4. Cell bodies near the tritocerebrum and sube-sophageal ganglion could be the same cells as those ob-served in per-gal4 transgenics (see above). However, neu-ronal PER immunoreactivity has never been observed inthis region (e.g., Siwicki et al., 1988; Ewer et al., 1992).Moreover, the TIM signal in this ventral brain region wasweak and appeared to be temporally constant within agiven day (data not shown), in contrast to the gross fea-tures of TIM cycling in head extracts (e.g., Myers et al.,1996; Zeng et al., 1996).

Reported per and tim expression in larvalbrains: axonal projections from putative

precursors of imaginal pacemaker neurons

per-gal4-driven marker expression. GAL4 expres-sion patterns in the larval CNS in three per-gal4 lines (1b,2, and 3) were studied in combination with UAS-gfp. CNSsfrom third-instar larvae (L3) carrying two copies of UAS-tau (line 24/1) and one copy of per-gal4 (line 2) were alsostudied by immunofluorescent staining using anti-TAU.All brains were observed by confocal microscopy in whole-mounted samples. Each of the three per-gal4 lines thathad similar GAL4-mediated reporter expression in adultsalso gave essentially identical expression patterns in thelarval brains. The TAU signals are shown in Figure 6Aand B.

These patterns are similar to those observed in trans-genics carrying a per-lacZ fusion transgene (Kaneko etal., 1997) in the following respects: larval LN cells werestained in most samples (31 of 35 brain hemispheresexamined; Fig. 6A,B). TIM/PER-immunoreactive larvaldorsal neurons-2 (DN2Ls, L for larval) were often diffi-cult to distinguish from the other dorsally located neu-rons, and they could be identified in only eight of the 36hemispheres examined by their position just anterior tothe pedunculus of the larval mushroom body. No DN1L

cell was found in any of these 36 hemispheres. In theventral region of the brain lobes, numerous cells werelabeled by GFP or anti-TAU (Fig. 6A). In a dorsolateralregion of the brain, a cluster of cells gave GFP- orTAU-immunostained signals (Fig. 6B). The only differ-ence from the expression pattern of the per-lacZ fusiongene is that GFP and TAU signals were found at thesurface of the CNS in 15 of 18 brains from three per-gal4lines examined. These signals are most likely glial cells


located over the surface of the entire brain (Fig. 6A, B;cf. Ito et al., 1995). The numbers of stained LN cellsvaried depending on the methods used to detect per-controlled GAL4 expression. For example, 4.2 6 0.5 LNswere recognized in larvae carrying two copies of UAS-tau and one of per-gal4, whereas 1.2 6 0.2 LNs were

labeled by GFP in larvae carrying one copy each ofUAS-gfp and per-gal4.

Ventral ganglia from 12 per-gal4 larvae were examinedfor marker expression. Cells near the midline at the ven-tral side of the ventral ganglia were labeled by GFP orTAU (Fig. 6A).

Fig. 5. Reported tim expression in the adult brain. A: Frontal viewof a wholemounted brain carrying tim-gal4 (line 86) and UAS-gfp (onecopy each); 26 confocal sections at 5.04-mm depth intervals are super-imposed; d, LNd cells; lv, large LNvs; sv, small LNvs (these three cellbody types are TIM-immunoreactive in wild-type); triangles, GFP-expressing neuronal cells in which TIM is normally undetectable; AL,antennal lobe (whose signals may come from nearby cells dorsolateralto the AL); Me, medulla optic lobe. B: GFP signals driven by tim-gal4(same genotype as in A); 27 5.04-mm confocal sections were superim-posed; Ol, optic lobe complex; Eso, esophagus. C: Higher magnifica-tion of right hemisphere of brain shown in B; 63 sections at 2.16-mmintervals are superimposed; 1, neurites of DN1 cells; d, neurites ofLNd’s; POT, neurites of large LNvs in the posterior optic tract; trian-gles, fibers appearing to originate from descending neurons near thesubesophageal ganglion. D: Horizontal view of dorsal brain from a flycarrying tim-gal4 (line 62) and UAS-gfp (one copy each); 53 2.16-mm

sections are superimposed; 1 with arrow, DN1 cell bodies; 1 witharrowhead, dorsal commissure originating from DN1s. E: Horizontalsection of a brain carrying one copy each of tim-gal4 (line 86) andUAS-tau (line 7); the TAU-immunostained section is at the level of theellipsoid body (EB) and the fan-shaped body (FB) in the centralcomplex; that structure is not stained, although glial cells surround-ing the EB and FB are; brown staining throughout the cortex mainlycomes from glial cells; R, retina. F: TAU expression in a section ca. 40mm ventral to the one in E (same fly); sv, 1, fibers from the small LNvsand DN1s that connect the dorsal protocerebrum and the region of LNcell bodies; Ol, optic lobe complex. G: TAU expression in a section ca.40 mm ventral to the one in F; AL, faintly labeled neuronal processesin the antennal lobe. For abbreviations, see list. Scale bar 5 50 mm inA (applies to A,B); 100 mm in C (applies to C,D); 100 mm in E (appliesto E–G).


tim-gal4-driven marker expression. With respect totim expression in the larval CNS, 10 lines of tim-gal4 werestudied in combination with UAS-gfp. Two of these (62and 82) were also studied in combination with UAS-tau(line 7). In all lines studied, expression of GFP, TAU, orboth was detected in all the TIM-immunoreactive cellclusters previously observed in wild-type larvae (cf.Kankeo et al., 1997): LN, DN2L, and DN1L (Fig. 6C–F;Table 5).

Because relatively small numbers of larval brain cellswere labeled, fibers from these neurons were readily trace-able. LNs initially project posteriorly to the larval opticneuropil where the larval photoreceptor axons terminate.Then these fibers turn anteriorly and dorsally and termi-nate near the cell bodies of the DN2Ls, which are locatedjust anterior to the pedunculus (Fig. 6D–F). The neuritepatterns just described are essentially the same as thosedetermined previously by application of anti-PDH to lar-val CNS specimens (Kaneko et al., 1997; Helfrich-Forster,1997).

In the tim-gal4 larvae, dendritic processes of LN cellswere labeled by GFP or TAU in the optic neuropil (Fig.6D–F). DN2Ls were observed to project medially, cross themidline, and terminate shortly after they enter the con-tralateral hemisphere (Fig. 6D–F). DN1Ls project posteri-orly to the brain region in which the LN axons terminate(Fig. 6D–F). Whether the DN1L neurites terminate in aregion lateral to the mushroom body pedunculus or extendfurther down, along with LN fibers, was not clear. Thiswas because of the extensive arborization observed in ourpreparations in these brain regions. To examine this mat-ter further, GFP expression was observed in larvae carry-ing tim-gal4 (line 27) and UAS-gfp along with a discon-nected (disco1) mutation. Such mutant larvae lack PDH-positive LN cells (Helfrich-Forster, 1997). By includingdisco1 in the genetic background of the transgenic larvae,GFP-marked projections from DN1L cells were revealed toterminate lateral to the mushroom body pedunculus,where the neurites of LNs end in wild-type (data notshown). However, whether DN1Ls in wild type still fascic-ulate with LN fibers to innervate the larval optic neuropilwas ambiguous. This is because the DN1Ls of larvae car-rying this mutation, which affects projections of visualsystem and those of other sensory cells that innervate theCNS (e.g., Glossop and Shepherd, 1998), may not be ableto project their axons in a normal manner.

Within the region of strongly labeled tim-expressingLNs (ca. five such neurons), approximately seven cellswith weak marker expression were recognized (Fig. 6E, F;Table 6). These additional signals most likely correspondto cells near the larval LNs that express weakly nativePER and TIM proteins (Kaneko et al., 1997). From some ofthese neurons with the faint reporter signals, TAU-labeled processes running medioanteriorly were traced in20 of 38 brain hemispheres examined (Fig. 6F). However,these neurite signals were rarely detected in samples ex-pressing GFP (two of 56 brain hemispheres). The neuronsgiving rise to these processes are reminiscent of adultLNds in terms of their cell body locations and projectionpatterns (see above).

There was an additional set of neurites in the larvaltim-gal4 specimens. These fibers were observed to ema-nate from some of the weakly labeled cells that are locatednear the strongly labeled LNs. Neurites as well asperikarya of the additional lateral cells were weakly la-beled and observed to fasciculate with neurites of LNs perse. The latter exhibit more intense marker expression andcourse posteriorly to the larval optic neuropil. From thisregion, the weakly labeled, laterally originating fibersmay project to the region of DN2L cell bodies along withthe strongly labeled neurites. However, weakly labeledfibers were also seen to run relatively straight from theoptic lobe neuropil toward the esophagus. Such neuriteswere recognized in eight of 10 tim-gal4 lines and in 69 of94 brain hemispheres examined (Fig. 6E,F). These fibersare analogous to the POT in the adult brain by location,although they extended all the way to the esophagus inonly two of 47 larval samples. The POT seems to bepresent in late embryos (Nassif et al., 1998). Therefore, ifthe weakly labeled projections from tim-expressing lateralneurons contribute to the larval POT, they would be sim-ilar to axons stemming from the large LNv cells in theadult brain.

Novel tim-expressing cells. Larval brain cells thathave not been recognized by TIM immunohistochemistry(Kaneko et al., 1997) were also found in several tim-gal4reporter-containing animals. In each of 62 brain hemi-spheres examined from seven transgenic lines, one neuronlocated medial and posterior to the DN2L cluster waslabeled (Fig. 6C,D,E). In the other three lines, such a cellwas not detected. This neuron sends processes medially tothe contralateral side as well as laterally to the regionwhere LN projections terminate. More posteriorly, an-other dorsally located neuron was labeled in 44 of 56 brainhemispheres examined from six lines (Fig. 6C,D,E). Itsends an ipsilateral projection to a region anterior towhere fibers emanate from DN2L cells. In the other fourlines, this cell was not detected. Weakly labeled cells werefound in a posterodorsal region in 60 of 86 brain hemi-spheres examined from seven lines (Fig. 6D,E); in theother three lines, these cells were not detected. In theventral region of the brain, one to seven weakly labeledcells were recognized in 69 of 91 brain hemispheres fromall 10 lines. The positions and numbers of the weaklylabeled cells in posterodorsal and ventral regions of thelarval brain varied, and no processes from these cells werelabeled. One tim-gal4 line gave signals in the optic stalk,another in a posteriorly located neuron that sends anipsilateral projection; these were not stained in larvae ofthe other strains. The four components of the reporterexpression patterns just indicated are very likely the re-

TABLE 5. Adult Brains Labeled by Anti-TAU or GFP Fluorescence, Drivenby tim-gal4, for Each Type of tim-Expressing Neuron and Their Fibers1,2

No. brainsobserved

No.labeled

Cell BodiesLarge LNv 40 40Small LNv 31 30LNd 40 37DN1 34 29DN3 29 17

FibersPOT 17 17Small LNv/DN1 17 8LNd 42 22DN1 commissure 38 29

1 Of 47 brains examined in toto, 12 were from two tim-gal4 lines (62 and 82) that alsocarried UAS-tau, and 35 brains were from four tim-gal4 lines that also carried UAS-gfp.2 For each type of tim-expressing neuron and their projections, numbers of brainsobserved are given in the second column. Numbers of brains in which a given neuronalcluster or structure was recognized are in the third column.


Fig. 6. Reported per and tim expressions in the larval centralnervous system (CNS). These images are from wholemounts of theentire CNS or the brain of third-instar larvae (L3). A: Entire CNS(ventral view) of L3 carrying one copy of per-gal4 (line 2) and twocopies of UAS-tau (line 24/1), stained by application of anti-TAU; 25confocal sections at 5.04-mm depth intervals are superimposed; LN,cell bodies of lateral neurons; Glia, glial cells at the surface of thebrain hemispheres; VG, ventral ganglia. B: Dorsal brain hemisphereswith per-controlled TAU expression (same genotype as in A); 592.16-mm sections were superimposed. C: Entire CNS of L3 carryingtim-gal4 (line 62) and UAS-gfp (one copy each), monitored for GFP-mediated fluorescence; 18 5.04-mm sections were superimposed; LN,lateral neurons, 1s with white arrows, DN1L cells; 2s with whitearrows, DN2Ls (these three cell body types are TIM-immunoreactivein wild-type); green arrows, cells not identified as TIM-containing,including signals near the dorsal brain midline (green 1s) and cellsmedial and dorsal to LN processes (green 2s); RG, ring gland; Gut,anterior region of larval alimentary system. D: High magnification ofdorsal brain hemispheres carrying tim-gal4 (line 86) and UAS-gfp(one copy each); 45 2.16-mm sections are superimposed; LN, cell bodiesof lateral neurons; 1,2 with with white arrows, cell bodies of DN1Ls

and DN2Ls, respectively; LN with arrowhead, fibers projecting fromlateral neurons; 2 with arrowhead, fibers projecting from DN2Ls;other labeling (green and RG) as in C. E: Dorsal brain hemispheresfrom an L3 carrying one copy each of tim-gal4 (line 62) and UAS-tau(line 7); 52 2.16-mm sections are superimposed; fiber patterns, espe-cially dendritic ones, are visualized better by expression of TAU (cf. C,D); small arrowhead, dendritic fiber of DN1Ls within the left hemi-sphere; small white arrows, POT-like fibers, putatively originatingfrom weakly labeled LNs (see F); other labeling as in C and D;asterisk, debris. F: Diagram of TAU marking in a tim-gal4 L3 brain(shown in E), depicting positions of known TIM-expressing neuronalsomata and their projections; color coding for cell bodies and theirneurites: light blue, strongly labeled LNs; green, weakly labeled LNs(putative precursors of LNd cells); purple, weakly labeled LNs (puta-tive precursors of large LNvs and their POT projections); pink, DN2Ls;dark blue, DN1Ls; because cell bodies and fibers from strongly labeledLNs, LNd-like cells, and large LNv-like ones overlap in the lateralbrain, drawings for the weakly labeled precursors of LNds and of largeLNvs are shown in the left hemisphere; the strongly labeled LNsappear on the right. For other abbreviations, see list. Scale bar 100mm in A 5 (applies to A,C); 50 mm in B (applies to B,D–F).


sult of position effects of the tim-gal4 transgenes’ chromo-somal locations.

In the ventral ganglia, GFP-labeled cells (mean: 3.9 60.4, n 5 38) were found in a lateral region within each sideof the brain in larvae from seven tim-gal4 lines (Fig. 6C).Similar numbers of cells in this part of the larval nervoussystem were found in TAU-labeled samples (data notshown). Two of the remaining three lines were assessed,and no signals were detected in this ventral CNS region.

Reported timeless expression duringpupal development

Products of the period gene become undetectable shortlyafter the onset of puparium formation in the two dorsallylocated neuronal clusters that contain per products inlarvae (Kaneko et al., 1997). Although per-expressing neu-ronal clusters are observed in a corresponding region inlate pupae and adults (Kaneko et al., 1997), it is not clearwhether these neurons arise late in development or in-stead correspond to the larval DN1L or DN2L cells.Marker expressions mediated by GAL4 are advantageousfor looking further into this matter, because some suchproteins have relatively long half-lives (Tissot et al., 1997;Li et al., 1998). Therefore, GFP expression patterns drivenby tim-gal4 were studied in early to mid-pupae.

tim-controlled GFP expression in early pupae.

Metamorphosing tim-gal4 animals from line 86 were ob-served for GFP signals, in specimens taken 0, 3, 6, and 9hours after puparium formation. Cells similar to all thenormal TIM-immunoreactive neuronal clusters seen inlarvae (LN, DN2L, and DN1L) were clearly recognized bystrong marking in all the brain hemispheres observed(between 30 and 44, depending on the number scrutinizedfor a given cell cluster). However, additional weakly la-beled cells were found in a dorsal region and a lateralregion of the brain (Fig. 7A, B). At these stages, expressionof a per-lacZ fusion gene product has been found only inLNs per se (Kaneko et al., 1997), not in the additionallaterally and dorsally located cells. Persistent tim-drivenGFP signals in DN2L- and DN1L-like neurons and in theirprocesses suggest that the apparent lack of per-lacZ ex-pression in the dorsal region is not due to degeneration oflarval DN2Ls and DN1Ls, but to naturally diminished perexpression in these cells after the onset of pupariation.

Similar to the results from larvae, weakly labeled LNsin pupal brains were recognized by tim-controlled GFPexpression. These cells may correspond to the large LNvsand LNds of adults, However, putative LNd processes wererevealed only in four of 30 brain hemispheres examined. A

fiber bundle continuous from one hemisphere to the con-tralateral side was observed in 15 of 22 pupal samples.This is similar to the POT in adult brains.

In addition to these dorsal and lateral clusters of pupalcells, which appear to correspond to larval structures, afurther cluster of GFP-labeled cells was recognized inspecimens of early pupae. Such signals were within abrain region corresponding to that of the adult DN3 cellsand were observed in 31 of the 32 hemispheres examinedfrom 0- to 9-hour pupae.

After 18 to 22 hours into pupal development, tim-expressing, GFP-labeled, dorsally located cell clustersfrom larvae begin to intermingle with the other cells thatexpressed this reporter in tim-gal4 animals (line 86). How-ever, DN2L- and DN1L-like cells were still recognized inthese brains by the location of somata, projection patterns,and intense GFP expression (Fig. 7C). In all of the sevensamples observed, DN2L-like cells were recognized bytheir locations near the terminals of LNs. Similarly,DN1L-like cells were recognized in these samples by theircell bodies, which are located slightly medial to the otherdorsal cells, and by their fibers, which run laterally to theregion where LN projections terminate (Fig. 7C). DN2L-like neurites in the anterior brain appeared to be thickerin 18- to 22-hour pupae compared with larvae and earlier-stage pupae (Fig. 7C). GFP-labeled neurites of neighbor-ing cells in dorsal brain regions appeared to take the samepathway as those of DN2L fibers.

In the ventral brain of 18- to 22-hour pupae, newlyemerging lateral neuronal signals were observed. Four orfive such cells were clearly labeled and located anterior tothe positions of small LNv cells, which are present inlarvae and persist into adulthood. The projections of neu-rites from the newly appearing cells were recognized infour of seven samples. Judging from their location, theseneurons most likely correspond to the large LNv cells inadults. It was difficult to distinguish putative small LNvsfrom large LNv precursors by sizes of the somata (Fig. 7C),possibly because the large LNv precursors are not yet fullydifferentiated in pupae. Varicose fibers observed to ar-borize into the developing medulla (Fig. 7C) are likely tooriginate from the maturing large LNv cells. However,PDH-immunoreactive fibers in the medulla, projectingfrom the large LNvs, have not been detected until 60% ofpupal development has progressed (Helfrich-Forster,1997). A further group of clock gene-expressing lateralneurons emerges during relatively late stages of develop-ment. These are the dorsal LN cells. LNd-like cells andtheir fibers were observed in four of seven brain hemi-spheres taken from 18- to 22-hour tim-gal4 pupae (Fig.7C). Labeling of such presumptive LNds was clearer com-pared to what was observed in larvae or 0- to 9-hourpupae.

With regard to dorsal neurons that appear to be theprecursors of DN3s in adults, such cells were observed in18- to 22-hour pupae in a region near the lateral horn (cf.Strausfeld, 1976). During this stage of metamorphosis,DN3-like cells appeared to express the tim-gal4 weakly, assurmised by low-level GFP reporting (Fig. 7C).

Reported tim expression in midpupae. Transgenicscarrying the UAS-gfp marker were examined approxi-mately halfway through pupal development. Between 36and 46 hours after puparium formation, DN2L- and DN1L-like cells were not readily recognizable. This was becausemany other cells in this dorsal brain region express the

TABLE 6. Larval Central Nervous System (CNS) Cells Expressing GFP orTAU Under the Control of tim-gal4 in TIM-Containing Cell Clusters

No.hemispheres

observed1No.

cells2

Cell typeLN 93 4.8 6 0.0Putative LNd and large LNv 92 7.1 6 0.2DN1L 94 2.0 6 0.0DN2L 94 1.9 6 0.0

1 Of 47 brains examined in toto, 19 were from two tim-gal4 lines (62 and 82) that alsocarried UAS-tau, and 28 brains were from 10 tim-gal4 lines that also carried UAS-gfp.Expression patterns in the TIM-expressing cells were similar in all the lines.2 Numbers of GFP- or TAU-labeled cells (6S.E.M.) are given in the third column foreach cluster of TIM-expressing neurons; S.E.M. values smaller than 0.05 are desig-nated as 0.0.


tim-controlled reporter. Yet, DN2L-like cells located closeto the terminals of LNs were observed in four of fivesamples from 36-hour but not in 46-hour pupae (Fig. 7D).DN1L-like neurons were found in three of five samplesfrom 36-hour pupae, and in one of three from specimenstaken at 46 hours (Fig. 7D). At these later stages, a com-missure like that emanating from DN2L cells in the dorsalbrain of larvae is even more extensive than was observedin 18- to 22-hour pupae (Fig. 7D).

Compared with the appearance of POT in larvae andearlier-stage pupae, this track was easily traced from oneside of the brain to the other in seven of eight samplesfrom 36- and 46-hour pupae (Fig. 7D). In such maturinganimals, the varicose fibers in the medulla that originatefrom the precursors of the large LNvs were more extensivecompared with 18- to 22-hour pupae (Fig. 7D). LNd-likecells and their processes were labeled in all eight samplesfrom 36- to 46-hour pupae (Fig. 7D). Fibers from these

cells make a loop near the cell bodies, like that observedfor LNd cells in adult brains. As in metamorphosing ani-mals from relatively early stages, the cell body size of thelarge LNv precursors was indistinguishable from that ofsmall LNvs, and DN3-like cells were weakly labeled (Fig.7D).

Reported per- and tim-expression innervous systems mutated for the

cycle or Clock genes

GFP expressions driven by per-gal4 and tim-gal4 wereexamined in larval and adult brains of animals homozy-gous for cycle01 (cyc01) or ClockJrk(ClkJrk) mutations.These cause eclosion and adult locomotor behavior to bearrhythmic and lead to very low levels of per and timproducts, as assessed in tissue homogenates (Rutila et al.,1998; Allada et al., 1998).

Fig. 7. Reported tim expression in the pupal brain. These pupaecarried tim-gal4 (line 86) and UAS-gfp (one copy each); for eachwholemounted brain, 46–64 confocal sections at 2.16-mm-depth inter-vals were superimposed. A: Three-hour pupa, in which the GFPpattern does not differ from that observed in larval brains; whitearrows (labeled as in Fig. 6D), cells at similar locations and withsimilar projection patterns as known TIM-expressing neurons in theL3 brain; 2 with arrowhead, fibers of DN2L-like neurons cf. Fig. 6D);triangles, non-TIM-immunoreactive cells; unlabeled white arrow-heads, fibers that may give rise to the posterior optic tract. B: Nine-hour pupa, in which the GFP pattern is again similar to that of L3,except that weakly labeled cells near the positions of LN and DNclusters within the larval brain exhibit stronger fluorescence at thispupal stage; structure labeling as in A. C: Brain hemisphere from a22-hour pupa (lateral is to the left); positions of GFP-expressing cellbodies and projection patterns are similar to those driven by tim-gal4

in adults; sv, small LNv somata; lv, large LNv cell body; d with arrow,LNd cell body; v, neurites of ventrolateral neurons; d with arrowhead,neurites of LNds (which could not be traced to their putative terminalregions); 1s with arrows, cells in dorsal brain region likely correspond-ing to that of DN1s in adult (among these cells, two nearest themidline are similar to DN1Ls of larva according to their position andprojection patterns); 2 with arrow, two overlapping somata (in aposition similar to those of L3 DN2Ls and adult DN2s); 1,2 witharrowheads, fibers appearing to project from several cells in the DN1region and to follow the same apparent pathway as that of L3 DN2L

projections; 3, putative precursor of DN3 cluster. D: Brain hemi-sphere from a 36-hour pupa (essentially the same region as in C);structure labeling as in C, with the LNd cluster (d) more prominentlylabeled compared to 22-hour pupae; POT, putative posterior optictract originating from large LNvs (lv). For other abbreviations, seelist. Scale bar 5 50 mm in A (applies to A–D).


Neuronal marking driven by per-gal4 in cyc01 and

ClkJrk adults. GFP expression in cell bodies of mostper-expressing neurons under the control of per-gal4 wasdetected in the face of homozygosity for either mutation(Table 7). However, the numbers of GFP-labeled DN3swere clearly reduced in the mutants compared with ani-mals carrying no clock mutations (Table 7). In suchcyc1Clk1 transgenic flies, marker gene-positive cells thatnormally do not contain TIM protein were found near thelocations of large and small LNvs, LNds, and DN1s thatare TIM-immunoreactive (Table 4). Therefore, it is notclear whether the small LNv-, LNd- and DN1-like cellsmarked by GFP in cyc01 and ClkJrk are true clock gene-expressing cells. Fibers from small LNvs and DN1s thatrun between the LN region and the superior protocere-brum were not detected in the mutant transgenics, imply-ing a reduction of GAL4 expression in these two per-expressing clusters. In contrast, fibers from the large LNvsin the POT as well as in the medulla were recognized inthe mutants (Table 7). Therefore, this cluster of neuronsseems unaffected by either cyc01 or ClkJrk. GFP signals incell bodies near the antennal lobes and their neurites,which are not labeled by anti-PER but commonly observedin per-gal4 strains (see above), were also unaffected by thetwo clock mutations (Table 7).

Neuronal marking driven by tim-gal4 in cyc01 and

ClkJrk adults. In both mutant backgrounds, GFP ex-pression in the cell bodies of large LNvs and in DN1s wasdetected, whereas fibers from the large LNvs in the POTas well as in the medulla were unaffected (Table 8). Re-ported tim expression in cell bodies of the small LNvsseemed present in both mutant backgrounds (Table 8),although these signals were not easily distinguished fromglial cells in the optic lobes. However, GFP signals in mostglial cells of the mutants were greatly reduced comparedwith those observed in cyc1Clk1 transgenics. The onlyexception involved glial cells at the distal margin of the

medulla, in which the reporter’s signal strength was un-affected by either mutation (Table 8). Fibers from DN3swere not detected in flies homozygous for cyc01 or ClkJrk

(Table 8). GFP signals driven by tim-gal4 in locationspreviously not labeled by anti-TIM in the adult brain, suchas cells near the antennal lobes and their projections, werenot affected by either mutation (Table 8). Marker expres-sion in the ellipsoid body, which was weak in nonmutantflies, was more readily detectable in cyc01- or ClkJrk-bearing flies compared to cyc1 Clk1 (data not shown). Thisanomaly could be the result of intrinsically higher expres-sion of the fluorescent marker in the mutant backgrounds.Alternatively, it may stem from lower than normal GFPsignals in glia (see above), which could unmask the EBsignal.

Certain differences between the effects of these twomutations on tim expression were found: signals in fibersfrom the small LNvs and the DN1s that run between theLN region and the superior protocerebrum, as well as thecommissure from the DN1s, were absent in most ClkJrk

specimens but present in cyc01 ones. Yet, GFP signalsin the DN1 commissure was clearly reduced in cyc01

compared to wild-type. Cell bodies of LNds and DN3swere detected in cyc01 but not in most ClkJrk individuals(Table 8).

Larval CNS marking driven by per-gal4 and tim-gal4

in cyc01 and ClkJrk mutants. GFP signals mediated byper-gal4 (line 2) or tim-gal4 (line 86) were examined forthe effects of these mutations on the developing nervoussystem. In both genetic backgrounds, GFP expression inLN cells within the brain, driven by per-gal4, was barely

TABLE 7. Cells and Fibers Expressing GFP Under the Control of per-gal4in cycle01 or ClockJrk Genetic Backgrounds1,2

Genetic backgroundNo. brains labeled/no. observed3

WT cycle01 ClockJrk

per-expressing neurons (cell bodies)Large LNv 4/4 6/6 6/6Small LNv 2/3 5/5 4/5LNd 4/4 5/5 5/6DN1 4/4 6/6 6/6DN3 4/4 3/54 2/64

per-expressing neuronal fibersPOT 4/4 5/5 3/3Medulla 4/4 6/6 6/6DN1/small LNv 3/3 0/4 0/4Dorsal commissure 4/4 0/4 0/4

GFP-labeled brain neurons (cellbodies) not expressing PER in WTPars intercerebralis 4/4 6/6 6/6Cells dorsal to AL (EB cells) 4/4 6/6 6/6Cells ventral to AL (mACT) 4/4 6/6 6/6TRI/SEG 4/4 6/6 6/6

GFP-labeled brain neurons (fibers)not expressing PER in WTCentral complex 4/4 6/6 6/6mACT 4/4 6/6 6/6

1 Four brains were observed for wild-type (WT), six for each of the cycle01 and ClockJrk

genetic backgrounds.2 Abbreviations as in Table 3.3 For all cells and fibers, the numbers of brains labeled are given as numerators,numbers of samples observed as denominators.4 Numbers of stained cells were reduced compared to wild-type.

TABLE 8. Cells and Fibers Expressing GFP Under the Control of tim-gal4in cycle01 or ClockJrk Genetic Backgrounds1,2,3

Genetic backgroundNo. brains labeled/no. observed3

WT cycle01 ClockJrk

tim-expressing neurons or glia (cellbodies)Large LNv 4/4 7/7 6/6Small LNv 4/4 7/76 4/56

LNd 4/4 7/7 1/6DN1 4/4 7/7 5/5DN3 1/15 7/7 0/6Glia (medulla) 4/4 7/7 6/6Glia (others) 4/4 7/77 6/67

tim-expressing neuronal fibersPOT 4/4 7/7 5/5Medulla 4/4 7/7 6/6DN1/small LNv 2/2 7/7 0/5LNd fiber 2/2 2/7 0/6Dorsal commissure 3/3 7/77 1/5DN3 fiber 2/2 0/7 0/5

GFP-labeled brain neurons (cellbodies) not expressing TIM in WTCells dorsal to AL 4/4 7/7 6/6TRI/SEG 4/4 7/7 6/6

GFP-labeled brain neurons (fibers)not expressing TIM in WTAL 4/4 7/7 5/5EB 4/4 7/7 6/6SEG fiber4 2/2 5/6 5/6

1 Four brains were observed for wild-type (WT), seven for cycle01, and six for ClockJrk.2 Abbreviations as in Table 3; medulla in the top section of the current table is thesecond-order optic lobe.3 For all cells and fibers, numbers of brains labeled are given as numerators, numbersof samples observed as denominators.4 Putative descending axon in the subesophogeal ganglion (SEG).5 In three of four samples, glial signals masked the DN3 signals.6 Small LNv signals detected could be glial, although their relatively large size androunded shape imply that they are neurons.7 Staining intensity was reduced compared to wild-type.


detectable compared with clock-normal controls (data notshown, obtained from one cyc01 and from three ClkJrk

larvae). Marker signals mediated by per-gal4 in DNL cellswere not reliably detected in a normal genetic background(see above), nor were these dorsal cells marked in themutant larvae. GFP marking driven by tim-gal4 was notdetectable in larval LNs, DN1Ls, or DN2Ls under theinfluence of either of these clock mutations (data notshown, obtained from three cyc01 and three ClkJrk larvae).In contrast, GFP signals mediated by per-gal4 in ventraland dorsolateral brain regions and in the ventral gangliawere not affected by cyc01 or ClkJrk. In these CNS loca-tions, PER immunoreactivity is low in wild-type (Kanekoet al., 1997). GFP expression mediated by tim-gal4 wasnot reduced for marker-positive cells in which TIM immu-noreactivity has not been detected in wild-type larvae. Forinstance, GFP expression in the ventral ganglion was notaffected by the clock mutations.

DISCUSSION

Whole-body expression patternsof clock genes

Application of tim-gal4 transgenes, driving a GFPmarker, showed that expression of the timeless clock geneis very widespread in larval tissues and especially those ofadults. This fits with the picture that emerged earlier forspatial expression of the period gene (Hall, 1995). It isnotable in this regard that the clock genes more recentlycloned in rodents, including orthologs of per and tim, allexhibit very broad distribution among a host of mamma-lian tissues and organ systems (reviewed by Dunlap,1999). However, we found that per and tim are not coex-pressed throughout their distribution in Drosophila. If thefunction of these genes has chronobiological significancein most tissues where the products are found, PER andTIM proteins would not be able to cooperate in terms ofmediating circadian pacemaker activity. Yet, this is theconventional view of how PER and TIM function, for ex-ample, to interfere with the positive transcription factorsencoded by cycle and Clock (Dunlap, 1999).

Neuronal expression of clock genes in cellbodies and their neurites

The period and timeless clock factors are coexpressed inmany regions within the CNS, although the expressionpatterns of these genes are not contiguous throughouttheir neural distributions. Most of the relevant data comefrom the current application of per-gal4 and tim-gal4transgenes (also see Hunter-Ensor et al., 1996; Kaneko etal., 1997; Stanewsky et al., 1998). We now discuss what isknown about per and tim-expressing neurons, includingtheir projections of neurites and developmental history. Asummary of these neuronal projection patterns is given inFigure 8.

Large ventrolateral neurons. This cluster of four tofive relatively large neurons is located at the anteriorinner margin of the medulla of the optic lobes (Fig. 8A).Their neurites in the posterior optic tract and the medullahave been studied in detail by using anti-PDH immuno-histochemistry (Helfrich-Forster and Homberg, 1993;Helfrich-Forster, 1995). We confirm this pattern in thepresent study (purple in Fig. 8A). Developmentally, bothPDH immunoreactivity and per expression have been ob-

served initially in midpupae (Helfrich-Forster, 1997;Kaneko et al., 1997). Marker signals driven by tim-gal4suggest, however, that the precursors of large LNv cellsare present even earlier during larval development andthat they persist through metamorphosis.

Small ventrolateral neurons. This is another later-ally located neuronal cluster consisting of five neurons.Their neurites projecting to the superior protocerebrumhave been revealed by anti-PDH immunostaining(Helfrich-Forster and Homberg, 1993; Helfrich-Forster,1995), which we confirm (light blue in Fig. 8A). These cellsbegin to express the per and pigment-dispersing factor(pdf) genes during the first-instar larval stage (Helfrich-Forster, 1997; Park et al., 2000). TIM protein is alsoknown to be in these cells at least in third-instar larvae(Kaneko et al., 1997). Expression of per and pdf as well asthe neurites of these cells persist throughout metamor-phosis (Helfrich-Forster, 1997; Kaneko et al., 1997; Parket al., 2000). The present data are consistent with theseprevious observations on the development of the smallLNv cells.

Dorsolateral neurons. This cluster consists of ca. sixneurons located dorsal to the other LN clusters in theanterior lateral cortex. The current study shows that mostof these cells coexpress per and tim. Their neurites, whichwere partially traced in this study, project dorsally to thesuperior protocerebrum (green in Fig. 8A). The onset ofper expression in this cluster was previously detected inmidpupae (Kaneko et al., 1997). Marker gene expressiondriven by tim-gal4 implies that these cells are in factpresent at least from the larval stage.

Dorsal neurons, group 1. The larval two-cell clustercalled DN1L is located within an anterodorsal region ofthe brain (Fig. 8A) and expresses both PER and TIM(Kaneko et al., 1997). These cells project posteriorly to theregion where LNs terminate and cell bodies of anotherdorsal cluster, called DN2L, are located (dark blue in Fig.8A). Notwithstanding the apparent termini of the DN1L

cells, it is possible that these neurites project farther pos-teriorly to the larval optic neuropil. Expression of the pergene in DN1Ls becomes undetectable just after the pupa-rium formation (Kaneko et al., 1997). The persistence oftim-gal4-driven GFP expression in this cluster in earlypupae indicates that this disappearance of per products isnot due to programmed cell death. Given that manylarval-specific neurons die during the first 24 hours afterpupariation (Truman et al., 1993), the larval DN1L cellsthat survive the first 20 hours of metamorphosis may beamong the brain neurons that persist throughout this latedevelopmental stage. Such cells could give rise to subsetsof the DN1 cluster in adult brains by turning on perexpression during later stages of pupal development.

The DN1 cluster in adults consists of ca. 15 neurons inthe dorsalmost cortex. Fibers from many of these neuronsseem to make a V-shaped commissure in the superiorprotocerebrum (dark blue in Fig. 8A). At least some ofthese neurons also project laterally and ventrally alongwith fibers from the small LNv cells. A few of the DN1smay project ventrally along the posterior surface of thebrain to the esophageal canal, then to the corpus cardia-cum. Although tim expression in the DN1Ls could persistthroughout metamorphosis to become subsets of DN1 cellsin adults, many other such neurons begin to express perand tim during pupal development (Kaneko et al., 1997;and the current study).


Fig. 8. Diagrams summarizing the development of wiring patternsfor clock gene-expressing neurons and putative output pathways fromcircadian pacemaker cells. A: Brains from three different stages; colorcodings for neurons expressing the period and timeless genes areindicated alongside the larval diagram as well as between the pupaland adult ones; cell bodies are depicted as circles; the numbers ofthese for most cell clusters are approximately the same as in thespecimens observed (e.g., Fig. 2B,D; Fig. 6F); however, the exactnumbers of cells within LNd- and large LNv-like clusters that expressper and tim weakly in the larval brain are not known because of lowlevels of endogenous PER and TIM and of tim-gal4-mediated markerexpression; also, circle numbers are fewer than the cell body countsfor DN1 and DN3 clusters in pupal and adult brains, because the realnumbers are so large (in adults, ca. 15 and 40, respectively); lineswithout arrowheads indicate neurites judged to terminate wherethese diagrammed tracings end; lines with filled arrowheads repre-sent neurites whose terminal endings could not be identified; thosewith open arrowheads represent neurites judge to be still developing;for the larval brain, LN’s strongly expressing PER and TIM are shownin the right hemisphere; LNd- and large LNv-like cells with weak PERand TIM immunoreactivities are in the left larval hemisphere; simi-larly in the pupal and adult diagrams, the small LNv and DN3 somataare shown only in the right hemisphere, LNd and large LNv somataonly in the left hemisphere; the purple projections are shown bothipsi- and contralaterally, because it is not clear whether large LNvssend neurites to the medulla optic lobe on one or both sides of thebrain; DN1s and DN2s are represented by circles and lines with the

same colors as their putative larval precursors, because similar pro-jection patterns as those DN1Ls and DN2Ls were identified in pupaeand adults (Figs. 2, 6, 7); separate DN1 cells seem to have differentprojection patterns, and the dark blue pathways are in part hypothet-ical: some DN1 fibers may terminate near the midline, but certain ofthese neurites cross it; others make a loop in the lateral protocere-brum, then project back to the midline; still other DN1s seem toproject ventrally to the esophagus (open oval); both ipsi- and con-tralateral DN1 projections to the optic lobes are shown, because thesepatterns could not be traced definitively. B: Possible output pathwaysof clock neurons in adults; CC, corpus cardiacum; PI and TRI, cells inthe pars intercerebralis and the tritocerebrum, along with their pro-jections (brown dashed lines); pathways from each clock gene-expressing neuronal type employ the same color scheme as in A; solidcolored arrows depict a hypothetical neuronal pathways that starts inthe key pacemaker cells, the small LNvs, and ends with rhythmicbehavior: thus, the light blue arrows connecting with DN1s, DN2s, orboth; these dorsal neurons send signals to the PI, which in turncommunicates with the CC, the thoracic ganglion (black arrows), orboth (see text within Discussion); LNds and DN3s, which project to thedorsal protocerebrum (green and red arrows), may also interact withDN1s, DN2s, or the PI and thus contribute to behaviorally meaningfuloutput; a coupling pathway putatively connecting contralateral LNs,and one that may involve communication between small LNvs andDN1s, are indicated by thick dashed arrows; a hypothetically directhumoral pathway from the brain to behavior is represented by a grayarrow, as is a humoral output from the CC. For abbreviations, see list.


Dorsal neurons, group 2. This DN2L group of cells inlarvae consists of two neurons located in a dorsal region ofthe brain just anterior to the pedunculus of the mushroombody (Fig. 8A). These neurons coexpress per and tim(Kaneko et al., 1997). The DN2L cells project medially andterminate just after they cross the midline (pink in Fig.8A). As in DN1Ls, per-expression in DN2Ls becomes un-detectable after the onset of pupariation (Kaneko et al.,1997). DN2L cells were detected from the beginning of thepupal development and for the next 20 hours by tim-gal4-driven GFP expression. Therefore, the DN2Ls do not un-dergo programmed cell death during early pupal develop-ment and may persist through metamorphosis to give riseto the DN2s of adults by turning per expression back on.Neurites from the adult DN1 cells seem to take the samepathway as those of the DN2Ls to give rise to the DN1commissure.

The DN2 cluster in adults also consists of two cellssituated just above the calyces of the mushroom body (Fig.8A). The similarity between positions of DN2s and DN2Lssuggests that they may be the same cells. Neurites fromthe DN2 cells could not be traced by marker gene expres-sion driven by per-gal4 or tim-gal4.

Dorsal neurons, group 3. This newly identified clus-ter consists of ca. 40 cells in the dorsolateral cortex lateralto the lateral horn (Fig. 8A). These neurons project medi-ally just below the DN1 commissure and terminate in thepars intercerebralis. Expression of per in the DN3s wasfirst detected in mid pupae (Kaneko et al., 1997). Thepresent study revealed tim-controlled GFP marking ofthem in early pupae.

Among the salient findings in this study are that mostclasses of rhythm-related neurons seem to exist even atthe larval stage. Moreover, the neuronal wiring patternsof these developing cells are similar to those in adults.Therefore, projections from larval clock neurons could bepioneer fibers that contribute to the formation of certainclock gene-expressing neurons within the adult (Fig. 8A).In the context of these rhythm-related larval cells, whichprefigure those in the adult brain, it was interesting toreveal separate onset times for the period and timelessgenes’ expressions. Thus the temporal patterns of theseclock genes activities, at least as reported by markers inthe gal4 transgenics, do not reveal the full developmentalhistory of such putative pacemaker cells. That is, we areobserving onsets of cellular differentiation as opposed tothe times when these neurons first exist.

Advantages of the GAL4-expression systemfor neuroanatomical studies and subcellular

localization of marker proteins

We used the GAL4 expression system to visualize thewiring patterns of clock gene-expressing neurons. PER,TIM, PER-bGAL fusion proteins are predominantly local-ized in nuclei and cell bodies because of their nuclearlocalization signals (see Introduction). Transgenes con-taining only per-regulatory sequences and marker genessuch as lacZ or luciferase-encoding sequences lead to insitu signals that are either too weak (Cooper et al., 1994),are located in nuclei due to an artificially added nuclearlocalization signal (for bGAL: Hao et al., 1999), or arelocalized predominantly within only one subcellular com-partment (which for luciferase is non-nuclear; Stanewskyet al., 1997b).

Flies carrying a transgene in which the per promoterwas ligated to sequences encoding a fusion protein of thekinesin motor protein and bGAL (cf. Giniger et al., 1993)were generated as a possible approach for visualizing theneuronal processes of clock cells in the Drosophila brain.Expression of this fusion protein did not reveal the pro-jection patterns of clock neurons. The signals consisted ofscattered spots throughout the brain that seemed to re-flect localization of the marker in terminals of such neu-rons, per-expressing glia, or both (C. Brandes, B. Frisch,and J.C. Hall, unpublished observation). Therefore, weneeded to devise a way to express markers that would beexpressed both in perikarya, for basic cell identification,and throughout the cells’ processes, to maximize visibilityof all the cells’ anatomical features.

The GAL4-expression system is suitable for this pur-pose for two reasons: (1) translated GAL4 levels result inamplification of gene expression driven by a given pro-moter (e.g., Johnson et al., 1995; Morimura et al., 1996);and (2) the binary nature of this system, involving combi-nations of the designed construct with UAS fusions, en-ables expression of different marker genes with differentsubcellular localization, all under the control of the samepromoter. Indeed, the GAL4 system has been used previ-ously to visualize neuronal pathways in Drosophila (e.g.,Yang et al., 1995; Shepherd and Smith, 1996; Smith andShepherd, 1996; Ito et al., 1997a, b; Tissot et al., 1997,1998).

We mainly applied two markers, GFP and TAU, al-though bGAL was used in some cases. GFP and TAUallowed neurite projections to be traced from most classesof clock neurons, at least partway between the cell bodiesand nerve termini. We submit that comparisons of resultsobtained with these two markers, and by augmenting thewholemount approach with the use of sectioned materialin certain cases, may have minimized problems that couldstem from efficiency of detecting labels in different prep-arations. For example, potential uncertainties associatedwith antibody penetration among different specimens of agiven genotype are belied by the small standard errorsthat accompany the cell counts in Table 4.

However, neuronal endings of some of these cells, suchas those projecting from LNds and DN1s, could not beidentified. This was partly due to superimposition of neu-rites from several clock gene-expressing cells, which inter-fered with the resolution of individual structures. In othercases, the neurite signals were too faint to trace fully,implying weak expression of GAL4 in those cells. Anotherfactor responsible for incomplete tracing of certain fibersmay be uneven distribution of marker gene products.Among the three markers, only GFP labels nuclei. This isbecause molecules less than ca. 60 kDa, such as GFP, candiffuse into the nucleus (e.g., Perez-Terzic et al., 1997).TAU seems to label the processes of cells better than dothe other two markers. For instance, in sectioned mate-rial, TAU revealed the fibers in the ellipsoid body and thelateral triangle better than did bGAL (Fig. 2I). Therefore,it is important to apply a combination of marker genes toreveal the entirety of GAL4-expressing cells. Still, thepresynaptic or postsynaptic nature of the nerve endingscould not be definitively determined in this study. Amarker gene whose product is transported actively to thepresynaptic terminals, such as a fusion protein of neuro-nal synaptobrevin and GFP (cf. Ito et al., 1998; Blagburnet al., 1999), could be combined with per-gal4 or tim-gal4


to gain further anatomical information of the fly’s rhythmsystem.

Unexpected expression patterns of markergenes controlled by per-gal4 or tim-gal4

In the transgenic strains carrying clock gene-gal4 fu-sions, marker signals were observed in unexpected tissuesand cells, compared to the spatial distribution of per andtim products in wild-type larvae and flies. Several featuresof the anomalous expression patterns in the transgenicsare likely to reflect the action of per and tim regulatorysequences (discussed later in this section). But other com-ponents of the ectopic marker expressions were not incommon among transgenic strains of a given type (e.g.,per-gal4). These kinds of signals are likely to be the resultof position effects associated with certain of the trans-genes’ chromosomal insertion sites. Such effects are ex-pected, because “enhancer trapping” is common for Dro-sophila transgenes containing GAL4- or reporter-encodingsequences (e.g., Wilson et al., 1990; Kaiser, 1993). Almostnone of the chromosomal sites in such transposon-taggingstudies has been characterized further, but it assumedthat these loci include regulatory sequences that can drivea given pattern of tissue expression.

Our transgene approach unwittingly encountered en-hancer trapping in some strains, but was based on well-characterized gene regulatory sequences as opposed to themere trapping of gal4 by unknown ones. Yet we assumethat in some of our per-gal4 and tim-gal4 strains, themarker signals were driven both by clock gene regulatorysequences and by an enhancer trapped at the transgene’schromosome-insertion site. Nevertheless, many of the re-porter signals observed in novel locations were in commonamong several strains of a given type (e.g., at least threeof the six per-gal4s). These patterns are unlikely to rep-resent ectopic expression that resulted solely from posi-tion effects of the transgenes’ chromosomal insertions.The ectopic marker locations that were commonly ob-served among several strains of a transgenic type includedbrain cells that have not been labeled by application ofanti-PER or by expression of PER-bGAL fusion protein(reviewed by Helfrich-Forster, 1996; Kaneko, 1998). Wepresume that the clock gene promoters were genuinelyactive in these “extra” CNS cells, compared to an expla-nation based on coincidental trappings of neural enhancerthat give the same spatial patterns among different trans-gene insertion sites. Thus, for example, the per regulatoryregion would be active in these ectopic locations, but nei-ther PER nor PER-bGAL fusion protein normally accumu-late in such cells. In contrast, signals mediated by TAU,GFP, and bGAL in the current set of transgenics couldaccumulate, because none of these marker proteins con-tains PER sequences that can target such material fordegradation. This hypothesis, involving transcription of aclock gene in certain cells and the relative instability ofthe final products within them, is elaborated in neuroana-tomical detail below. If it is correct, then the gene’s mRNAwould be detected in such locations. A potentially relevantstudy was performed on wild-type brains, in which thespatial distribution of per RNA was determined by in situhybridization (Rachidi et al., 1997). However, such signalscannot be compared with the ectopic reporter-mediatedones found in the current study, because Rachidi et al.(1997) did not study the central brain regions relevant tothe current study in detail.

We now compare the marker patterns mediated by per-gal4 and tim-gal4, focusing on the genuine signals thatare not the result of position effects and enhancer trap-pings. To visualize clock neurons in adults, per-gal4 wasmore useful than tim-gal4, whereas tim-gal4 providedbetter visualization of clock neurons in the developingnervous system. The patterns controlled by tim-gal4 werenot very useful for adults because of the strong expressionof marker genes in numerous glial cells. Lack of glialsignals in most of the per-gal4 lines was unexpected, be-cause PER protein is found in many glia (e.g., Siwicki etal., 1988; Ewer et al., 1992; Helfrich-Forster, 1995). Theabsence of glial expression of per-gal4 in multiple linesmay reflect the activity of the regulatory region used inthis study. The ca. 4-kb upstream per sequence used hasbeen applied several times, for example, in a per-luciferasefusion transgene called plo-luc (Brandes et al., 1996;Plautz et al., 1997b; Stanewsky et al., 1997b). An analo-gous reporter construct, called NbG, contains the 4-kbupstream sequence, the 59-untranslated region of per’ssecond exon, and lacZ (Zwiebel et al., 1991; Cooper et al.,1994). Both types of fusion genes lack not only the codingsequence of per but also its large first intron. Overalllevels of expression of both constructs were low comparedwith the reporter signals stemming from constructs inwhich the same 4-kb upstream region drives expression ofPER-bGAL or PER-LUC fusion proteins (Cooper et al.,1994; Stanewsky et al., 1997b). The expression pattern ofNbG in glia could not be determined because of weak anddiffuse staining obtained by X-gal histochemistry (Cooperet al., 1994), and plo-luc expression was low in glia(Stanewsky et al., 1997b). The weak expression of report-ers driven by per-gal4 in the eye may also be due toinadequacy of the per regulatory sequences included inthis construct.

In order to express GAL4 in a pattern similar to thepatterns determined for endogenous PER, sequencesdownstream of the gene’s promoter region, such as thosewithin per’s first intron, may be necessary. In fact, regu-latory elements downstream of the transcription-start siteseem to be important for normal kinetics of per transcriptoscillation, the full spatial expression pattern, and dosagecompensation of this x-chromosomal gene (Cooper et al.,1994; Frisch et al., 1994; Stanewsky et al., 1997b; So andRosbash, 1997). However, a 69-base pair (bp) sequencepresent in the 4-kb sequence was shown to mediate ex-pression of a fused lacZ gene in a per-like pattern, includ-ing glial and photoreceptor expression of the reporter (Haoet al., 1999). A critical difference between the currentstudy and that of Hao et al. (1999) may involve a heterol-ogous basal promoter and a nuclear localization signal,which were positioned between the 69-bp sequence andthe lacZ reporter by Hao et al. (1999). In contrast, nopromoter sequences other than those of per were includedin our per-gal4 construct.

Transgenic flies carrying tim-gal4 were especially use-ful for revealing clock neurons in developing animals,because of their restricted patterns of expression com-pared with those of per-gal4 strains. This difference in thedeveloping CNS reflects the endogenous gene expressionof per and tim. PER-bGAL fusion protein has been foundin neurons of dorsolateral clusters within the brain lobes,in the ventral brain, and in the thoracic-abdominal CNS,where endogenous PER expression is very low (Kaneko etal., 1997). TIM immunoreactivity in these cells was not


detected, suggesting that the tim gene may not be ex-pressed there (Kaneko et al., 1997). In the absence of TIM,PER accumulates to anomalously low levels (Price et al.,1995). This is consistent with the low levels of PER proteinobserved in the TIM-negative, PER-bGAL-expressingcells just mentioned. Anomalously elevated PER can occurin another genetic context. Thus, PER in the larval CNScells under discussion was observed in a double-time (dbt)mutant but not in wild-type (Price et al., 1998). The dbtgene encodes a relative of human casein-kinase Ie (Klosset al., 1998), which seems in Drosophila to be involved inthe PER degradation that occurs normally in these larvalneurons. Our observations of stable GFP and TAU in thedorsolateral cluster and ventral regions of the brain lobes,as well as in the ventral ganglia, are analogous to thepersistent presence of PER in these same neural regions ofdbt mutant larvae (Price et al., 1998).

In the CNS of tim-gal4 larvae, cells were found thathave not previously been identified as TIM-immuno-reactive. These cells were observed in several lines oftim-gal4, indicating that the in situ signals reflect pro-moter activity of the 6-kb timeless upstream sequenceused in this study. Similarly in the adult brain, markerexpressions in cells previously unknown to express per ortim were found in several strains of per-gal4 or tim-gal4.Although endogenous expression of per or tim has notbeen detected in these cells (Siwicki et al., 1988; Ewer etal., 1992; Hunter-Ensor et al., 1996; Kaneko et al., 1997),real promoter activities of per or tim are likely to occur insuch locations. Their detection would be permitted only bythe application of reporters or direct detection of mRNAs(cf. Rachidi et al., 1997).

In summary, the following kinds of mechanisms mayunderlie differences between the wild-type and transgenicpatterns: one or more factors necessary for the stability ofendogenous proteins may not be expressed in cells whereonly marker gene expression was found, not endogenousPER or TIM. Alternatively, expression of GFP and TAUmay be delayed, due to the binary nature of the reportersystem. These reporter gene products could persist longer,due to the stability of the marker proteins compared withendogenous PER or TIM. Certain previous findingsprompt the explanations just given. For example, TAUsignals in flies carrying a GAL4 enhancer trap elementand UAS-tau were found to be delayed compared withlacZ expression in flies carrying the same GAL4 elementand UAS-lacZ (Tissot et al., 1997; Stocker et al., 1997).This suggests that TAU is more stable than bGAL. There-fore, some of the GFP or TAU signals detected in our studymay properly reflect the per or tim expression that wasinitiated during earlier developmental stages. Indeed, peris expressed not only in the brain of embryos but also inmany neurons within the ventral region of thoracic andabdominal segments (Liu et al., 1988; Siwicki et al., 1988).In the larval CNS, native PER expression in the ventralganglion becomes very weak (Kaneko et al., 1997). Thestrong TAU and GFP signals in the ventral ganglion oflarvae carrying per-gal4 and UAS-gfp or UAS-tau could bean example of anomalous persistence of reporter proteinsinto this postembryonic stage. A similar argument couldexplain the marking of certain neurons in the thoracicnervous system of adults, which was unexpectedly ob-served in the current study. A previous one concluded thatper is expressed only in glia within the thoracic and ab-dominal ganglia of adults (Ewer et al., 1992). Whereas

neuronal signals in these ganglia might have been missedamong the numerous glial cells expressing the clock genein this part of the adult CNS, an alternative explanation isthat the neuronal expression of marker genes reflectsper-promoter activity. Given that PER is found in neuronswithin the ventral ganglia of larvae (Kaneko et al., 1997;Price et al., 1998), stable marker proteins may persist intoadulthood even if per and per-gal4 are not transcribed inthose imaginal cells.

Effects of clock mutations on expression ofthe period and timeless genes

The cycle and Clock mutations could be expected nearlyto eliminate the expression of period and timeless, as de-tected in situ. This is because cyc01 and ClkJrk each causegross severe depressions of per and tim products (Allada etal., 1998; Rutila et al., 1998). However, both tim-gal4- andper-gal4-mediated marker expressions were spared in ap-preciable numbers of brain cells, in transgenics homozy-gous for either mutation. These results can be explainedby the following: Levels of per and tim mRNA levels andthose of both encoded proteins are greatly reduced intissue extracts, but not zero in these mutant backgrounds(Allada et al., 1998; Rutila et al., 1998). This allows for thepossibility that residual amounts of these gene productsare concentrated in a subset of the spatial expressionpatterns. Moreover, amplification of per- and tim-promoter-mediated marker gene expression by GAL4maximized the detectability of cells expressing per andtim weakly in these mutants. In any case, the current datashow that marker gene expressions in some of the per- andtim-expressing cells were greatly reduced by these clockgene mutations, whereas signals in other cells were notapparently affected. Such a selective elimination of perand tim expression by cyc01 or ClkJrk mutation impliesthat factors other than CYC and CLK are involved inexpression of per and tim in some brain cells.

Roles played by clock neurons other thansmall LNv cells in the control of

behavioral rhythms

Among all the cells in the adult brain that express per,tim, or both, the small LNvs are considered to be impor-tant pacemaker cells that are necessary and sufficient forthe generation of robust circadian behavioral rhythms inconstant condition (Helfrich-Forster, 1996; Kaneko, 1998).Fibers of the small LNvs project dorsally and terminate inthe superior protocerebrum, first revealed by PDH immu-noreactivity (Helfrich-Forster and Homberg, 1993) andconfirmed in the current study. A correlative study of PDHimmunoreactivity and adult behavioral rhythms of discoflies indicated that these fibers in the superior protocere-brum are necessary for robust behavioral rhythms in con-stant darkness (DD), because rare mutant flies with PDH-positive neurons that failed to innervate the superiorprotocerebrum were not rhythmic (Helfrich-Forster,1998). However, other clock gene-expressing neurons maycontribute to behavioral rhythmicity, especially in light-dark (LD) cycling conditions, because all disco individualssynchronize to LD cycles (Dushay et al., 1989; Hardin etal., 1992; Wheeler et al., 1993), even though most aredevoid of LN cells. Furthermore, many disco animals arerhythmic for first few days in DD, and a few such fliessustain their rhythmicity for over a week (Wheeler et al.,


1993; Helfrich-Forster, 1998). Though they largely lackLNs, the brains of disco adults retain per-expressing DNsin apparently normal numbers and positions (M. Kaneko,unpublished observations). Therefore, the DN cells may beresponsible for entrainment and could mediate ephemeralfree-running rhythmicity in DD.

We believe that the more dorsally located cluster of LNcells also contributes to locomotor circadian rhythms. Therare disco individuals that exhibit robust rhythmicity forseveral days in constant darkness, but are missing PDH-immunoreactive LNvs (Helfrich-Forster, 1998), may re-tain LNd cells. Such neurons not only express per and tim(Stanewsky et al., 1998), but they also send neurites intothe dorsal brain that terminate near to the locations ofLNv nerve terminals (Fig. 8). These dorsal LNs and theirneurites would be retained in adult brains that had allLNv cells specifically ablated by manipulation of the pdfgene (Renn et al., 1999). Such flies are frequently arrhyth-mic behaviorally (Renn et al., 1999) but not as severely asare the disco mutants (Dushay et al., 1989; Hardin et al.,1992; Helfrich-Forster, 1998), most individuals of whichlack all LN cells (Zerr et al., 1990). Therefore, comparingthe behavioral effects of pdf-mediated LNv ablations withthose of disco emphasizes the putative contribution tolocomotor rhythmicity of LNd and DN cells. The latter twotypes of neurons may interact, as LNvs and DNs arehypothesized to do below, because of the dorsal brainprojections that we now reveal for LNd neurites.

In this respect, one of the interesting current results isthat putative presynaptic terminals of small LNvs arelocated near the region where the cell bodies of DN2sreside. This is the location to which fibers from DN1 cellssend their immediate projections. Therefore, DN1 or DN2cells (DN1/2s) and the small LNvs may make functionalconnections with each other, such that the DN1/2s wouldbe postsynaptic to the small LNvs along the anatomicalpathway from the pacemaker cells to the generation ofbehavioral rhythms. This scheme is depicted in Figure 8B.Although robust oscillations of clock gene products in theDNs could be sufficient for the mere synchronization oflocomotor rhythms to LD cycles (see above), the smallLNvs may modulate the activity of DNs in order to gener-ate fully normal behavioral rhythms in LD conditions.Compatible with this suggestion, the LD entrainment ofdisco flies, which we postulate to be controlled by DN1/2cells, is evident but anomalous (Dushay et al., 1989; Har-din et al., 1992; Helfrich-Forster, 1998).

In constant darkness, DN cells would be unable to sus-tain behavioral rhythmicity in the absence of the signalfrom the small LNvs (see above). Under this hypothesis,the immediate postsynaptic target of the signal from thesmall LNvs to the DN1/2s may be a pacemaking mecha-nism operating in the DN1/2s. In this scenario, clock geneproduct oscillation in the DN1/2s would not undergorather rapid damping in the absence of LD cycles (cf.Wheeler et al., 1993). Alternatively, the small LNvs mayregulate only neuronal activity of the DN1/2s rather thanthe molecular oscillations in question. If DN1/2 cells arepostsynaptic to the small LNvs, which cells are targets ofthe DN1/2s? Certain neurites of those dorsal cells mayterminate near the midline at the pars intercerebralis(PI), as do the DN2Ls in the larval CNS (Fig. 8A). In thePI, there are many neurosecretory cells that arborize inthe CC or in the tritocerebrum (Fig. 8B, cf. Rajashekharand Singh, 1994). In the latter brain structure, dendritic

processes of certain descending neurons have been ob-served that innervate the thoracic nervous system (Rajas-hekhar and Singh, 1994).

Therefore, the following anatomical pathway for theadult behavioral rhythms is proposed (Fig. 8B): oscillatorysignals of the “master pacemaker” in the small LNvs firstmodulate the oscillatory mechanism or neuronal activityoperating within the DN1/2 cells. The DN1/2s send theiroscillatory signals to neurosecretory cells in the PI, whichmay lead to cycling level of humoral release in the CC aswell as in the tritocerebrum. Cyclic hormonal release inthe CC could therefore contribute to the control of behav-ioral rhythms (Fig. 8B). Alternatively, oscillatory informa-tion may be further transferred from the tritocerebrum tothe thoracic nervous system, which would lead to thenormal pattern of rhythmic motor output in LD conditions(Fig. 8B). In fact, involvement of the PI in circadian outputsystem is suggested in several insect species (reviewed inHelfrich-Forster et al., 1998). It is also conceivable thatthere is a direct humoral pathway from the brain to thebehavioral output (Fig. 8B), because transplanted brainsfrom donor flies carrying the perS mutation into the abdo-mens of per01 null-mutant flies were able to rescue behav-ioral rhythmicity of the latter, and the restored rhythmshad a short free-running period characteristic of the donortissue (Handler and Konopka, 1979).

Lastly, the small LNvs may not send inputs to the DNsalong a relatively simple, unidirectional output pathwayfrom clock cells to locomotor rhythmicity. Some of theDN1s seem to send fibers parallel to those projecting fromthe small LNvs and in the opposite direction (Fig. 8B).This apparent DN1 output heads toward the region of thelateral neurons. Therefore, DN1s may functionally con-nect with small LNv cells or their neurites (Fig. 8B). Thisadditional layer of putative functional complexity could beinvolved in the fine-tuning of behavioral rhythmicity. Forinstance, the LNd and DN3 cells, which project to thedorsal protocerebrum (Fig. 8A), may interact with cells inthe PI or the DN1/2 cluster and thereby modulate behav-iorally relevant outputs of the circadian clock (Fig. 8B).

In summary, most of the neurons in Drosophila thatexpress the period and timeless genes are interneuronal.None appears to be a motor neuron. The majority of thecells in which these clock genes are active, except therelatively large ventrolateral neurons, innervate the su-perior protocerebrum. Therefore, this dorsal region withinthe brain may receive rhythmic output from various clockneurons, integrate such signals, and convey such consoli-dated information to motor output in order to producecyclical oscillations of behavior.

ACKNOWLEDGMENTS

We thank S.P. Koushika for injection of the tim-gal4construct, K. Ito and S. Schneuwly for UAS-tau strains, B.Dickson for UAS-gfp, N. Perrimon for UAS-lacZ, and M.Rosbash for anti-TIM antiserum. We appreciate com-ments on the manuscript from J.D. Levine, J.E. Rutila,and K. White. We thank V. Shandalov for technical assis-tance and E. Dougherty for helping with confocal micros-copy and photography. This work was supported by agrant from the US NIH (GM-33205).


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Documents

Neuroanatomy of cells expressing clock genes inDrosophila: Transgenic manipulation of theperiod andtimeless genes to mark the perikarya of circadian pacemaker neurons and their projections