Institut de Neurobiologie Alfred Fessard, CNRS UPR 2216 (NGI), 91198 Gif-sur-Yvette, France
*Author for correspondence (e-mail: rouyer{at}iaf.cnrs-gif.fr)
Accepted 12 December 2001
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SUMMARY |
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Key words: Bolwig organ, Hofbauer-Buchner eyelet, Circadian clock, Rhodopsins, norpA, Dendritic tree
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INTRODUCTION |
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How do the Drosophila brain clock neurons see light? In contrast to mammals, whose eyes provide the only photic input to the clock located deep in the suprachiasmatic nucleus of the brain (Morin, 1994), flies appear to use several pathways for the light-resetting of their brain clock. The circadian clock of eyeless [e.g. glass (gl) or sine oculis (so)] or functionally blind [e.g. no receptor potential A (norpA) or transient receptor potential (trp)] mutants responds to light with a reduced sensitivity (Emery et al., 2000
; Helfrich-Förster et al., 2001
; Stanewsky et al., 1998
; Wheeler et al., 1993
; Yang et al., 1998
). This suggests that the visual system contributes to circadian photoreception but that other components are involved as well. The finding of a Drosophila gene (cry) encoding a blue light photoreceptor, cryptochrome, has revealed a new clock-specific photoreception pathway (Emery et al., 1998
; Stanewsky et al., 1998
). cryb mutants display defects in several clock responses to light (Stanewsky et al., 1998
) that can be rescued by targeted CRY expression in the LNvs, suggesting CRY-mediated light perception within the clock neurons themselves (Emery et al., 2000
). As expected, norpAP41; cryb double mutants were more affected than either simple mutants in their entrainment to light-dark (LD) cycles (Emery et al., 2000
; Stanewsky et al., 1998
). However, the double mutants still entrained, indicating that a third input pathway is used by the brain clock to perceive light in a norpA-independent manner (Hall, 2000
; Stanewsky et al., 1998
). This pathway appears to be glass dependent, as the clock that governs activity rhythms is completely blind in gl60J cryb double mutants (Helfrich-Forster et al., 2001
); it may rely on the Hofbauer-Buchner (HB) eyelet, a set of extra-retinal neurons that project into the anterior medulla of the adult brain, where the LNvs are located (Hall, 2000
; Helfrich-Forster et al., 2001
; Hofbauer and Buchner, 1989
; Yasuyama and Meinertzhagen, 1999
).
Although no clock-controlled behaviors have been characterized in larvae, larval clock function has been demonstrated by both molecular and behavioral studies (Kaneko et al., 2000; Kaneko et al., 1997
; Sehgal et al., 1992
). Sehgal et al. showed that the locomotor activity rhythm of the adults could be phased by a single 12-hour light episode during the first larval stage (Sehgal et al., 1992
). In another study, short light pulses given to entrained third-instar larvae were shown to shift the phase of the molecular rhythms of the larval LNs and of the adult activity rhythms (Kaneko et al., 2000
). Importantly, norpAp41; cryb double mutants were unable to entrain the molecular rhythms of their LNs to LD cycles delivered up to the third larval stage, suggesting that the larval light input pathways may be less redundant than in the adult, with a norpA-dependent visual system playing a more significant role (Kaneko et al., 2000
). The larval visual system consists of a pair of 12-cells organs, the Bolwig organs (BO). These organs express chaoptin, as retinal photoreceptors do, and send axonal projections (the Bolwig nerves or BNs) that enter into the brain via the optic stalks as early as embryonic stage 16 (Green et al., 1993
; Meinertzhagen and Hanson, 1993
). The BO has been shown to mediate several light-induced larval behaviors (Busto et al., 1999
; Hassan et al., 2000
). In contrast to the retinal photoreceptors, the larval ones are cholinergic rather than histaminergic (Yasuyama et al., 1995
), but seem to involve the same phototransduction cascade that uses rhodopsin(s) and norpA-encoded phospholipase C (PLC), according to the behavioral analysis of mutants (Busto et al., 1999
; Hassan et al., 2000
). Although their projections in the brain have not been extensively studied, they have been shown to terminate at the vicinity of the LNs, suggesting that the clock cells could be their direct targets (Kaneko et al., 1997
).
We report strong developmental interactions between the BN and the LNs, which start very early during development. In addition, we show that the disappearance of the chaoptin-expressing BN at the beginning of metamorphosis coincides with a remodeling of the LNs, and is followed within 1.5 days by the appearance of a new visual input to the LNs that comes from the neurons of the Hofbauer-Buchner eyelet underneath the retina. Interestingly, the photoreceptors of the adult eyelet and those of the Bolwig organ appear to express the same subset of rhodopsins, as well as the norpA-encoded PLC. The consequences of these findings for circadian photoreception are discussed.
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MATERIALS AND METHODS |
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Histology
Central nervous systems from third instar larvae, staged pupae and adults were dissected as described elsewhere (Blanchardon et al., 2001), except that primary antibody incubations were shortened to 4 hours at room temperature or overnight at 4°C, and the samples were preincubated with 10% normal goat serum before secondary antibody labeling. Embryos were dechorionated for 5 minutes with bleach on a plastic filter, rinsed and prefixed in an Eppendorf vial containing a 1:1 mix of heptane and 4% paraformaldehyde in phosphate-buffered saline (PBS). After the vial was turned slowly for 15 minutes, the lower (aqueous) phase was eliminated with a Pasteur pipette, and the embryos were collected in a small volume of heptane. They were forced on to a double-faced Scotch tape on the bottom of a small hollow dissection chamber filled with 4% paraformaldehyde in PBS, and the vitelline membrane was removed manually under a stereomicroscope. The embryos were then rinsed several times with PBS before proceeding with the standard immunofluorescence protocol (except that primary antibody concentrations were doubled). GFP was clearly visualized on live, dechorionated embryos, but it was lost during their further immunocytochemical processing. gal1118-driven UAS-lacZ expression was therefore used to detect the LNs in embryos. It was revealed with either a monoclonal anti-ß-galactosidase antibody or a rabbit anti-ß-galactosidase antiserum (for double-labeling experiments with mAb 24B10), which resulted in a higher background. Dilutions for the antibodies were as follows: mouse anti-chaoptin monoclonal antibody (mAb 24B10) (Fujita et al., 1982
), 1/100; mouse anti-ChAT monoclonal antibody (mAb 4B1) (Yasuyama et al., 1995
), 1/100; mouse anti-ß-galactosidase monoclonal antibody (Promega), 1/1000; rabbit anti-crab ß-PDF (Dircksen et al., 1987
), 1/5000; rabbit anti-NORPA (Zhu et al., 1993
), 1/5000; and rabbit anti-ß-galactosidase polyclonal antibody (gift from B. Limbourg-Bouchon), 1/100. Labeling of the somda and GMR-gal4/+; UAS-Kir 2.1/+ brains and their corresponding controls was performed with a newly generated rabbit anti-Drosophila-PDF (Neosystem, Strasbourg, France) with high specificity and low background, at 1/10000 dilution. Secondary antibodies were Texas Red or Alexa594-conjugated goat antibodies to rabbit IgG (Cappel or Molecular Probes, respectively), Texas Red- or Cy2-conjugated goat antibodies to mouse Ig (Cappel or Amersham, respectively). They were used at 1/1000 dilution, except for the Alexa594-conjugated goat antibodies (1/10000).
Imaging and image analysis
For measurements of the dendritic arborization of LNs, each mutant genotype was tested independently, in parallel with an identically treated wild-type control. Images were made from an epifluorescence microscope (Zeiss Axioplan2) with a cooled digital camera (Diagnostic Instruments SPOT2). For every half brain, the presence or absence of a GFP-stained dendritic arborization was scored (see Table 2). When it was present, its area in pixels was measured on the image, using a specific function of the SPOT2 software. The average area for a given genotype was normalized to that of the wild-type control in the same experiment, allowing comparison between the mutant strains. Confocal imaging was performed with a Leica TCS4D or SP2 confocal microscope.
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RESULTS |
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The eyes absent2 (eya2) mutation results in the absence of any adult photoreceptors (Bonini et al., 1993) without affecting the BN, which was thus the only visual structure stained in late third-instar larval brains (Fig. 3F). This situation is the opposite to that found for GMR-hid (see Fig. 3D), namely the absence of the BN and the presence of developing adult photoreceptors reaching into the brain. Contrary to GMR-hid, gl60J or somda, the dendritic arborization of the LNs in eya2 larvae appeared normal (Fig. 3F and Table 1), thus further confirming that the BN is specifically required for the presence of a wild-type dendritic arborization of the LNs.
Light-dependent BN activity is not required for normal morphological differentiation of the larval LNs
In an attempt to define how the larval optic nerve affects the dendritic arborization of its target LNs, we examined the role of light-driven BN activity by analyzing blind or dark-reared flies. The null norpAp24 mutation blocks the phototransduction cascade in the adult photoreceptors, and is likely to do so in the BN (Busto et al., 1999; Hassan et al., 2000
). The dendritic arborization of the LNs was of normal size in this mutant as it was in wild-type larvae reared in complete darkness throughout development (w DD, Table 1). These results show that the development of the dendritic tree of the LNs does not depend on the phototransduction cascade within the BN.
In order to test whether some light-independent activity of the BN might be involved, we expressed under GMR-gal4 control several molecules expected to alter or block BN function and analyzed their effect on the LNs. Tetanus-toxin light chain expression in the BN did not appear to affect the morphology of either the BN or the LNs (data not shown). However, expression of a potassium channel (KIR2.1) known to hyperpolarize neurons and strongly inhibit the firing of action potentials (Baines et al., 2001) led to alterations of both the nerve and its target arborization (Fig. 4A,B). These results suggest that light-independent activity of the BN is necessary and sufficient to induce and maintain the normal morphology of the dendritic arborization of the LNs. Interestingly, similar LNs defects were observed in a fraction of wild-type brains at the beginning of metamorphosis (Fig. 4C,D), when the BN might have begun to lose activity before it becomes undetectable with photoreceptor-specific markers.
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(1) The eyelet expresses a specific rhodopsin isoform, RH6 (Yasuyama and Meinertzhagen, 1999). Similarly, we found that the rh6-gfp construct strongly labeled the LNvs-contacting fibers in the adult brain (Fig. 7A,B). It allowed us to follow their long ventral course, alongside the PDF-expressing arborization, showing that the two structures had a much more extensive contact zone than could be deduced from sectioned material (Hofbauer and Buchner, 1989
; Yasuyama and Meinertzhagen, 1999
).
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(3) The HB cell bodies are located beneath the posterior retina (Hofbauer and Buchner, 1989; Robinow and White, 1991
; Yasuyama and Meinertzhagen, 1999
) and project toward the anterior medulla (Hofbauer and Buchner, 1989
). In order to visualize the LNs-contacting fibers together with the corresponding cell bodies in a wild-type context, we used a ro-tauZ construct that is co-expressed with rh6-gfp only in these fibers (data not shown). As expected for the eyelet, the ro-tauZ- labeled cell bodies were found immediately outside the distal margin of the lamina (Fig. 7I,J), with their fibers projecting to the anterior medulla (Fig. 7I), where they contacted the LNvs (not shown).
Phototransduction components within the eyelet
We then asked whether the eyelet would express the same phototransduction components than those expressed in the BO, in addition to RH6. NORPA expression was indeed detected in the eyelet of wild-type (Fig. 7K) and so1 flies (not shown). Although no RH5 was detected with a specific antibody, as previously described (Yasuyama and Meinertzhagen, 1999), we observed a weak expression of rh5-gal4 in the HB fibers (Fig. 7C,D), in 23% of dissected brain hemispheres. No labeling was seen with either rh3-gal4 or rh4-gal4 (not shown). These data indicate that the HB eyelet may use the same rhodopsins and phototransduction pathway as the BO.
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DISCUSSION |
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Precocious interaction between the BN and clock neurons
Differentiation of the BO starts at stage 12, and includes a multi-step elongation of the BN that pauses near the superficial optic lobe pioneer cells (OLPs), and finally reaches its target(s) inside the central brain at stages 16-17 (Campos et al., 1995; Green et al., 1993
; Schmucker et al., 1997
). The gal1118 enhancer-trap line (Blanchardon et al., 2001
) shows that the LNs are already present at embryonic stage 17. Their differentiation had probably occurred even earlier, as neuritic processes were already observed at that stage. Taking into account the various time lags introduced by the GAL4/UAS system, actual gal1118 expression in the LNs may similarly start as soon as or even before the BN reaches the central brain.
Use of gal1118-driven GFP fluorescence revealed that the dendritic tree of the LNs in third instar larvae is much larger than described with PDF immunocytochemistry and displays extensive intertwining with the BN ending. The presence of the acetylcholine-synthesizing enzyme ChAT in the terminal branches that contact the LNs strongly suggests that cholinergic synapses transmit light information from the BO to the clock cells, although additional transmitters are not excluded. Because the BN fasciculates with the axons of the OLPs (Campos et al., 1995), the latter may also contact the LNs. This possibility is strengthened by the fact that at least one of the OLPs is cholinergic (Yasuyama et al., 1995
).
Larvae carrying the norpAp24 mutation show defects in some light-induced behaviors (Busto et al., 1999; Hassan et al., 2000
), but the presence of the norpA-encoded PLC in the BO has not been documented before. Its detection down to the BN ending contrasts with the absence of detectable PLC immunoreactivity in the adult retinal axons (data not shown) (see also Fig. 7K) (McKay et al., 1995
; Zhu et al., 1993
). This different subcellular distribution could be due to the lack of specialized rhabdomeres in the BO cells (Green et al., 1993
). It could also reflect the expression of different PLC isozymes by alternative splicing from the norpA locus (Kim et al., 1995
; Zhu et al., 1993
). Expression of the retinal subtype I transcripts would be expected in the BO, but only body subtype II transcripts were reported in pre-adult stages (Kim et al., 1995
). Whether the subcellular targeting of the two PLC isozymes differs is not known. In any case, NORPA distribution all along the BN suggests that the relevant isozyme is involved in more than phototransduction.
This report indicates that the rh5 and rh6 genes are expressed in the BO but that rh1 is not. Similar data, as well as the absence of rh3 and rh4 expression, have been recently cited as unpublished results (Papatsenko et al., 2001). Interestingly, some larval responses to light have been reported to rely on RH1 (Busto et al., 1999
; Hassan et al., 2000
), suggesting a role for RH1 outside the BO. In the adult eye, RH5 and RH6 are found in different sets of R8 photoreceptors (Chou et al., 1996
; Huber et al., 1997
; Papatsenko et al., 1997
). Our data similarly suggest a mutually exclusive expression of the two types of rhodopsins in the larval photoreceptors, as RH5- and RH6-expressing fibers do not appear to overlap in the BN.
Visual afferences affect the differentiation of clock neurons
The ablation of the LNs did not induce morphological changes at the BN terminus. This contrasts with the strong effects that are often observed on a presynaptic neuron in the absence of its target (Campos et al., 1992; Sink and Whitington, 1991
), and may reflect the existence of other targets of the BN in this region (Mukhopadhyay and Campos, 1995
). However, the severe deficiency of the BN, in glass, GMR-hid or somda flies, had a drastic effect on the dendritic tree of the LNs. This demonstrates that the BN is required for proper morphogenesis of the LNs, and suggests that the BN is the main afferent connection to these clock cells. Interestingly, the BN is required also for the development of a serotonergic arborization that contacts its ending in late second instar larvae (Mukhopadhyay and Campos, 1995
). This contact suggests a serotonin-mediated modulation of BN-mediated light input to the larval brain clock. An inhibitory role of serotonergic afferents on retinal input to the mammalian suprachiasmatic nucleus has been well documented (Morin, 1999
), and is described for some effects of light on insect clocks (Cymborowski, 1998
).
Presynaptic nerve activity is often involved in the development or stability of postsynaptic elements (Cline, 2001). Our results point towards the involvement of some phototransduction-independent activity of the BN in the proper development of the dendritic arbor of clock cells. The disappearance of chaoptin expression in the BN at the beginning of metamorphosis correlates with a strong reduction of that arbor, which is also suggestive of a functional connection between the BO photoreceptors and the clock cells. The striking neuritic extension from the LNs that we observed in larvae expressing the KIR2.1 potassium channel in the BN has its counterpart in a small fraction of wild-type prepupae, consistent with the BN activity being altered at this developmental stage. Remodeling of dendritic arborizations during metamorphosis has been described for several subsets of larval neurons that persist into the adult stage (Tissot and Stocker, 2000
).
The HB eyelet differentiates concomitantly with and projects to the adult LNvs
Taken together, our anatomical and genetic data identify the adult LNvs-contacting photoreceptors as the HB eyelet (Hofbauer and Buchner, 1989; Robinow and White, 1991
; Yasuyama and Meinertzhagen, 1999
). As expected, their projections run close to the surface of the medulla to reach the anterior part of this neuropil, and they are present in the so1 mutant. In the wild type, the very extensive contact zone between these visual afferences and a PDF-expressing arborization closely matches the ventral extension of the accessory medulla, which was proposed as the target of visual inputs to the clock (Helfrich-Förster, 1997
).
During metamorphosis, the absence of chaoptin-expressing visual afferences to the LNs may last over 30 hours. Cryptochrome may thus be the only light input pathway to the clock during this time window. Chaoptin-expressing fibers contact the LNs again from 45 hours APF, at about the same time when the l-LNvs are first detected using pdf-gal4 as a marker. In the adult, the HB eyelet neurons appear to express both histamine and acetylcholine (Hofbauer and Buchner, 1989; Yasuyama and Meinertzhagen, 1999
). Whether both neurotransmitters are used for the light input to the adult LNvs, and if so, whether they target the small and large LNvs, or only one of the two groups, remains to be investigated.
Our observations are consistent with the report of three to six eyelet cells (Yasuyama and Meinertzhagen, 1999). The same report indicated that the eyelet expresses RH6 but not RH1, RH4 and RH5 rhodopsins. We too could not detect any anti-RH5 labeling, but weak rh5 expression was detected in the HB photoreceptors with a rh5-gal4 transgene, with most brains showing no rh5 expression. As mentioned above, rh5 and rh6 expressions are mutually exclusive in the retinal R8 cells, and our data suggest that the same rule may hold in the BO. In the retina, rh5 is expressed in only a minority of the R8 cells (Pichaud et al., 1999
). Similarly, rh5 could be expressed in only a minority of HB photoreceptors (and plausibly none in some eyelets, given the small number of cells). In any case, the low RH5 expression in the eyelet suggests that the relative contributions of RH5 and RH6 to circadian photoreception are different. These contributions could be tested by the analysis of circadian photoreception in specific rhodopsin mutants.
The possibility that the HB eyelet derives from the BO has been discussed in several studies. Despite differences in the number and position of cell bodies (Hofbauer and Buchner, 1989; Meinertzhagen and Hanson, 1993
; Yasuyama and Meinertzhagen, 1999
), and the 30 hours temporal gap between the disappearance of the BN and the detection of the eyelet (see above), recent results suggest that the eyelet cells may indeed be BO survivors (T. Edwards and I. A. Meinertzhagen, personal communication). This is in agreement with our finding that the BO and the eyelet appear to express the same phototransduction components. However, the presence of the HB eyelet in a few somda mutant adults, whereas the BN is never observed in larvae (Serikaku and OTousa, 1994
), would rather support a BO-independent origin for the eyelet. Alternatively, somda BO/eyelet precursors could be able to project into the brain, and enter their final differentiation program during metamorphosis, without prior embryonic differentiation as BO photoreceptors.
The HB eyelet has been proposed to be a circadian photoreceptive organ (Hofbauer and Buchner, 1989; Yasuyama and Meinertzhagen, 1999
). Our findings that its axonal projections directly contact the PDF-expressing arborization of the LNvs in the accessory medulla strongly support this hypothesis. How might the eyelet contribute to clock responses to light? Adult norpAp41; cryb double mutants still entrain to LD cycles (Emery et al., 2000
; Stanewsky et al., 1998
), while gl60J cryb double mutants do not (Helfrich-Forster et al., 2001
), suggesting the presence of glass-dependent, norpA- and cry-independent adult photoreceptors. Because the HB eyelet is absent in glass mutants, it appeared to be a candidate for such photoreceptors (Hall, 2000
; Helfrich-Förster et al., 2001
). Our finding that norpA is expressed in the eyelet strongly suggests that this structure actually participates to norpA-dependent circadian photoreception. PER-expressing dorsal neurons were recently shown to be missing in adult gl60J brains, making them alternative candidates for norpA-independent circadian photoreceptors (Hall, 2000
; Helfrich-Förster et al., 2001
).
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ACKNOWLEDGMENTS |
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