no receptor potential A

DEVELOPMENTAL BIOLOGY

Adult

The norpA gene encodes a 7.0-kilobase transcript that can be detected in the head but not in the body of adult flies and a 5.6-kilobase transcript that can be detected throughout development and in both heads and bodies of adults. In situ hybridization of cDNA sequences to tissue sections shows that the gene is expressed in the neuronal cell bodies of the optic lobe, central brain, and thoracic ganglia of adults and the brain of larvae. This tissue distribution of norpA transcripts is identical to the distribution of transcripts from a Drosophila Go alpha-subunit gene (Shortridge, 1991).

Antisera against NorpA recognize an eye-specific protein of 130-kDa relative molecular mass that is present in wild-type head extracts but not in those of strong NorpA mutants. The protein is associated with membranes and can be extracted with high salt. Immunohistochemical analysis at the light and electron microscopic levels indicates that the protein is expressed in all adult photoreceptor cells and specifically localized within the rhabdomeres, preferentially adjacent to, but not within, the rhabdomeric membranes. The results of the present study strongly support the previous suggestion that the norpA gene encodes the major phosphoinositol-specific phospholipase C in the photoreceptors. Moreover, insofar as the rhabdomeres are specialized structures for photoreception and phototransduction, specific localization of the NorpA protein within these structures, in close association with the membranes, is consistent with the proposal that it has an important role in phototransduction (Schneuwly, 1991).

Northern analysis, Western blots, phospholipase C activity assays, and immunohistochemical staining of tissues were all used to examine the tissue-specific expression of the norpA gene. It is expressed in a variety of tissues in addition to the eye. Hybridization of norpA cRNA probe to blots of poly(A+) RNA reveals that the gene encodes at least four transcripts: a 7.5-kilobase (kb) transcript that is expressed in eye and 6.5-, 5.5-, and 5.0-kb transcripts that are expressed in adult body or early stages of development. The 7.5-kb transcript is missing from eyes absent mutants and norpA mutants. Antiserum generated against the major gene product of norpA recognizes a 130-kDa protein that is abundant in eyes but severely reduced or absent in norpA mutants. The NorpA antiserum also recognizes a 130-kDa protein in adult legs, thorax, and male abdomen, but not female abdomen. These localizations are supported by results of phospholipase C activity assays that show that the norpA mutation reduces phospholipase C activity in each of the tissues in which NorpA protein can be detected. Furthermore, immunohistochemical staining of tissue sections with the NorpA antiserum demonstrates that the NorpA protein is abundant in the retina and ocelli and is present to a lesser extent in the brain and thoracic nervous system. Staining in the brain is strongest in the optic lobes and cerebrum. Since some of the above mentioned tissues that express NorpA (such as thorax, legs, and abdomen) have no known photoreceptor tissue, it has been concluded that the norpA gene product is also likely to have a role in signaling pathways other than phototransduction (Zhu, 1993).

Effects of Mutation or Deletion

Eight norpA mutants have been characterized by electroretinogram (ERG), Western, molecular, and in vitro PLC activity analyses. ERG responses of the mutants show allele-dependent reductions in amplitudes and retardation in kinetics. The mutants also exhibit allele-dependent reductions in in vitro PLC activity levels and greatly reduced or undetectable NorpA protein levels. Three carry a missense mutation and five carry a nonsense mutation within the norpA coding sequence. In missense mutants, the amino acid substitution occurs at residues highly conserved among PLCs. These substitutions reduce the levels of both the NorpA protein and the PLC activity, with the reduction in PLC activity being greater than can be accounted for simply by the reduction in protein. The effects of the mutations on the amount and activity of the protein are much greater than their effects on the ERG, suggesting an amplification of the transduction signal at the effector (NorpA) protein level. Transgenic flies were generated by germline transformation of a null norpA mutant using a P-element construct containing the wild-type norpA cDNA driven by the ninaE promoter. Transformed flies show rescue of the electrophysiological phenotype in R1-R6 photoreceptors, but not in R7 or R8. The degeneration phenotype of R1-R6 photoreceptors is also rescued (Pearn, 1996).

Mutations in the norpA gene of Drosophila severely affect the light-evoked photoreceptor potential with strong mutations rendering the fly blind. The norpA gene has been proposed to encode phosphatidylinositol-specific phospholipase C (PLC). A chimeric norpA minigene was constructed by placing the norpA cDNA behind an R1-6 photoreceptor cell-specific rhodopsin promoter. This minigene was transferred into norpAP24 mutants by P-element-mediated germline transformation to determine whether it could rescue the phototransduction defect concomitant with restoring PLC activity. Western blots of head homogenates stained with NorpA antiserum show that NorpA protein is restored in heads of transformed mutants. Moreover, transformants exhibit a large amount of measurable PLC activity in heads, whereas heads of norpAP24 mutants exhibit very little to none. Immunohistochemical staining of tissue sections using NorpA antiserum confirm that expression of NorpA protein in transformants localizes in the retina, more specifically in rhabdomeres of R1-6 photoreceptor cells, but not R7 or R8 photoreceptor cells. Furthermore, electrophysiological analyses reveal that transformants exhibit a restoration of light-evoked photoreceptor responses in R1-6 photoreceptor cells, but not in R7 or R8 photoreceptor cells. This is the strongest evidence thus far supporting the hypothesis that the norpA gene encodes phospholipase C, which is utilized in phototransduction (McKay, 1995).

The norpA (no receptor potential) mutant of Drosophila melanogaster has a visual transduction deficit. This study determines whether lack of function leads to structural repercussions in photoreceptor cells of the compound eye and their synapses. For this purpose, thin sections and freeze fracture replicas of norpA were examined using transmission electron microscopy. Ultrastructurally, retinula cells in the compound eye and all aspects of the first optic neuropil (lamina ganglionaris) are essentially normal in newly emerged flies. However, as expected, intraretinular pigment granules fail to show their light elicited aggregation; further, the P face particle density is somewhat lower than in wild type. There are unusual membrane specializations on the plasmalemma of the retinula cell, dubbed 'zippers'. Zippers appear to increase with age and can cause a distorted geometry of ommatidia. Only a few retinula cells ultimately degenerate in norpA, and the proportion may not differ from that of wild type. Despite the absence of the receptor potential in norpA, many aspects of the turnover of rhabdomeric membrane appear to be as in wild type (Stark, 1989).

The electrophysiological characteristics of norpAH52, a temperature sensitive phototransduction mutant of Drosophila melanogaster, were studied in vivo. Upon raising the environmental temperature to 33-37 degrees C, mutant flies exhibit time-dependent changes in photoresponses. Initial observations indicate losses in responsiveness at low light intensities and prolonged receptor potential waveforms. Subsequently, reductions in response amplitudes at higher light intensities occur, until no responses are obtained. On return to lower temperature the electrophysiological properties recover in reverse order. Based on these observations it is concluded that the primary defect of norpA affects the efficiency of the phototransduction process. Enhanced light exposure can offset the receptor potential changes in norpA. With the temperature sensitive mutant, (1) additional light exposure prolongs the time that responses can be observed at the higher temperature; (2) when 1-s illuminations no longer elicit responses at the higher temperature, 1-min illuminations at the same intensity temporarily restores the ability to obtain 1-s-responses, and (3) light accelerates the restoration of responses on return to lower temperatures. Illumination also has an effect on non-temperature sensitive norpA mutants, enabling the production of small photoresponses in norpAH44, a mutant that normally does not exhibit any responses, and improving the low-light-intensity responses of norpAP16. The current study indicates that the PI cycle, which is inhibited in norpA mutants (Yoshioka, 1985), is an important light-sensitive positive step or effector in the production of receptor potential responses (Wilson, 1987).

The norpAH44 phototransduction mutant of Drosophila, an allele that, on eclosion, does not exhibit a receptor potential was found, at later ages, to undergo light and temperature dependent degeneration of its photoreceptors as well as decreases in rhodopsin concentration. Pseudopupil measurements and light and electron microscopy were used to monitor the structure of the photoreceptors. When norpAH44 flies are maintained exclusively in the dark, no changes in structure or rhodopsin concentration are observed. When maintained on a 12 h light-12 h dark cycle, structural changes are first observed at 6 days of age for flies maintained at 24 degrees C or at 12 days of age for flies maintained at 19 degrees C. When the light-dark cycle is initiated after 10 days in the dark there is a more rapid loss of rhodopsin concentration and pseudopupil. The data suggest that even in the dark, although no obvious changes in structure or rhodopsin concentration are observed, certain processes that support these components have been affected. NorpAP12, an allele that exhibits small receptor potential amplitudes, also displays age- and light-dependent photoreceptor degeneration and decreases in rhodopsin concentration, whereas no degeneration or decreases in rhodopsin are observed in norpAP16, an allele that exhibits receptor potential amplitudes similar to those of wild-type. The data suggest that the processes that affect phototransduction, such as the phosphatidylinositol cycle, have a long-term role in the maintenance of rhodopsin concentration and photoreceptor integrity (Meyertholen, 1987).

The formation of stable rhodopsin-arrestin complexes induces apoptosis and photoreceptor cell degeneration

Although many different mutations in humans and Drosophila cause retinal degeneration, in most cases, a molecular mechanism for the degeneration has not been found. This study demonstrates the existence of stable, persistent complexes between rhodopsin and its regulatory protein arrestin in several different retinal degeneration mutants. Elimination of these rhodopsin-arrestin complexes by removing either rhodopsin or arrestin rescues the degeneration phenotype. Furthermore, it is shown that the accumulation of these complexes triggers apoptotic cell death and that the observed retinal degeneration requires the endocytic machinery. This suggests that the endocytosis of rhodopsin-arrestin complexes is a molecular mechanism for the initiation of retinal degeneration. It is proposed that an identical mechanism may be responsible for the pathology found in a subset of human retinal degenerative disorders (Alloway, 2000).

This paper demonstrates the existence of a novel mechanism to explain the light-dependent retinal degeneration that is observed in a subset of Drosophila visual system mutants. Mutations in three distinct genetic loci, norpA, arr2, and rdgB, result in the light-dependent formation of stable rhodopsin-arrestin complexes. Elimination of either member of this complex rescues the retinal degeneration in each of the three genetic backgrounds. In addition, it is shown that the formation of these stable rhodopsin-arrestin complexes triggers apoptotic cell death. Furthermore, endocytosis is essential for inducing cell death in norpA mutants, suggesting that the internalization of the rhodopsin-arrestin complexes is an early step in the initiation of apoptosis of retinal photoreceptors. It is possible that the excessive endocytosis saturates a downstream cellular function (for example, saturation of the early endosome or depletion of an endocytic protein), which signals the cell to undergo programmed cell death. Alternatively, the endocytosis may block a signal that protects the cell from apoptosis (Alloway, 2000)

The inactivation of the Drosophila phototransduction cascade is an extremely rapid event. Drosophila photoreceptors can shut off the light-activated currents in less than a 100 ms following termination of the light stimulus. One way in which photoreceptors have evolved to do this is at the level of the G protein-coupled receptor rhodopsin. Immediately upon activation, rhodopsin is multiply phosphorylated on its C terminus, and this phosphorylation greatly increases its affinity for the abundant soluble protein arrestin. Invertebrate Arr2 is a very basic molecule with a pK_a of ~8.7 and, therefore, has a high affinity for phosphorylated rhodopsin. Thus, this interaction of activated rhodopsin and arrestin occurs very rapidly, and the receptor is quickly inactivated. It is tempting to speculate that the posttranslational modifications of arrestin and rhodopsin are essential to eliminate these complexes. In such a model, it is necessary to phosphorylate Arr2, thereby making it less basic, as well as to dephosphorylate rhodopsin, thereby making it less acidic. Thus, the system is designed such that the influx of calcium activates CamKII, the kinase that phosphorylates arrestin, and rdgC, the phosphatase that dephosphorylates rhodopsin. Both of these steps are crucial, as evidenced by the fact that rdgC mutants and arr2(S366A) mutants both undergo rapid light-dependent retinal degeneration. Evidence suggests that, in the absence of these posttranslational modifications, stable rhodopsin-arrestin complexes persist in the cell and are instrumental in the pathology of retinal degeneration (Alloway, 2000)

The retinal degeneration induced by rhodopsin-arrestin complexes can be partially rescued by a temperature-sensitive allele of dynamin. This observation suggests that the rhodopsin-arrestin complexes are being removed from the photoreceptor rhabdomere by the endocytic machinery. A similar receptor internalization pathway occurs for the vertebrate β-adrenergic receptor. β-arrestin is involved in the inactivation and internalization of the β-adrenergic receptor. A small domain near the carboxyl terminus of β-arrestin directly interacts with clathrin and targets the β-adrenergic receptor-β-arrestin complex for internalization. This clathrin binding site is curiously missing in the visual arrestins. However, in spite of the absence of the clathrin-interacting motif, these complexes seem to have the ability to interact with the endocytic machinery. One attractive model is that visual arrestin serves as an AP2-like adaptor, just like β-arrestin, but recruits clathrin by a different mechanism. The question of whether the endocytic proteins are recognizing motifs in rhodopsin or arrestin awaits further studies. Many models have been proposed to explain retinal degeneration in humans, including constitutive activity of the phototransduction cascade, improper trafficking of photoreceptor cell components, and defects in the recycling of rhodopsin. The results described in this study implicate the endocytic pathway in retinal degeneration and suggest that excessive endocytosis can be a trigger for apoptotic cell death (Alloway, 2000)

The internalization of rhodopsin-arrestin complexes via receptor-mediated endocytosis could partly explain the membrane association of arrestin in certain mutant backgrounds. The enhanced membrane affinity of arrestin could in part be due to the rhodopsin-arrestin complexes rapidly interacting with the endocytic machinery. It is possible that, once the complex is associated with clathrin cages, the arrestin would be locked in the membrane-associated state. In support of this model, it has recently been demonstrated that the unphosphorylated form of arrestin directly interacts with clathrin in vitro. Therefore, it is possible that the role of arrestin phosphorylation is to block interactions with clathrin, and any mutant background that fails to phosphorylate arrestin yields complexes due to clathrin interactions. However, it is still essential that a stable rhodopsin-arrestin complex be formed initially to allow for the assembly of the endocytic proteins (Alloway, 2000)

A large number of mutations in the human rhodopsin gene have been isolated that are responsible for retinal disease. Interestingly, there are several mutations in human rhodopsin that form stable rhodopsin-arrestin complexes. These include a mutation in a highly conserved lysine in the seventh transmembrane domain that has been shown to cause autosomal-dominant retinitis pigmentosa. This mutant form of rhodopsin is found both in vivo and in vitro to be constitutively phosphorylated and tightly bound to arrestin. It had been hypothesized that the complexes between rhodopsin and arrestin may be instrumental in the retinal degeneration process. Possibly, as in Drosophila, these stable human rhodopsin-arrestin complexes are removed from the rod outer segments by the endocytic machinery, and this excessive endocytosis is the direct cause of apoptotic retinal degeneration (Alloway, 2000)

One question yet to be addressed is why photoreceptor cells have a mechanism for eliminating rhodopsin-arrestin complexes if, under nonpathological conditions, these complexes do not persist in the cell. One possible explanation is that arrestin has a secondary function to eliminate defective rhodopsin molecules from the photoreceptor cell. A rhodopsin molecule that becomes photochemically damaged may render the receptor nonfunctional or constitutively active. The presence of a constitutively active rhodopsin molecule could potentially be very detrimental and, therefore, necessitates its removal from the photoreceptor cell. Presumably, any rhodopsin molecule that becomes constitutively active will bind arrestin and generate a stable rhodopsin-arrestin complex. The stabily bound arrestin would target the dysfunctional rhodopsin molecule for endocytosis and degradation. In this manner, arrestin could function as a surveillance protein, eliminating defective rhodopsin molecules from the photoreceptor cell. However, in certain mutant backgrounds, the regulation of rhodopsin-arrestin complex formation is defective, and a large number of complexes are formed. The increased endocytosis of these complexes initiates apoptosis and retinal degeneration (Alloway, 2000).

Photoreceptor cells adapt to bright or continuous light, although the molecular mechanisms underlying this phenomenon are incompletely understood. This paper reports a mechanism of light adaptation in Drosophila, which is regulated by phosphoinositides (PIs). Light-dependent translocation of arrestin is defective in mutants that disrupt PI metabolism or trafficking. Arrestin binds to PIP(3) in vitro, and mutation of this site delays arrestin shuttling and results in defects in the termination of the light response, which is normally accelerated by prior exposure to light. Disruption of the arrestin/PI interaction also suppresses retinal degeneration caused by excessive endocytosis of rhodopsin/arrestin complexes. These findings indicate that light-dependent trafficking of arrestin is regulated by direct interaction with PIs and is required for light adaptation. Since phospholipase C activity is required for activation of Drosophila phototransduction, these data point to a dual role of PIs in phototransduction (Lee, 2003).

The demonstration that translocation of Arr2 is regulated by PIs addresses a lingering question concerning potential roles of PIs in photoreceptor cells. The Drosophila visual transduction cascade is among the most intensively studied GPCR cascades. During the last 30 years, many proteins and mutations have been identified that perturb PI signaling; however, the targets and mechanisms directly regulated by PIs have not been previously described (Lee, 2003).

The regulation of Arr2 shuttling by PIs occurs on the order of a few to many minutes. This is in contrast to the millisecond time scale, which operates in the activation of phototransduction. Although the specific activation mechanism involved in Drosophila phototransduction remains elusive, it is established that it depends on a PLCβ (NORPA). Thus, PLC-mediated hydrolysis of PIP₂ leads to rapid activation of the light-sensitive channels through the millisecond generation of PIP₂ metabolites or reduction in PIP₂ levels. Since adaptation occurs over a much slower timescale, regulation of this latter phenomenon exclusively by direct effects of second messengers on protein activities might be too rapid. Rather, regulation of adaptation by the translocation of signaling proteins provides a mechanism whereby changes in second messengers, such as PIP₃, result in delayed effects on the magnitude and the kinetics of signaling. Therefore, PIs appear to have the capacity to serve a dual role in activation and adaptation by modulating the activities and localization of signaling proteins (Lee, 2003).

Previous reports have shown that stable Arr2/rhodopsin complex formation leads to retinal degeneration in norpA or rdgC flies. Removal of the arr2 gene in a norpA or rdgC background partially suppresses the photoreceptor cell death. This partial suppression could be due to elimination of Arr2/rhodopsin complexes, reduction in endocytosis of rhodopsin, or disruption of some other Arr2 function. In this work, Arr2/rhodopsin binding and PI-regulated trafficking of Arr2 were uncoupled by expressing Arr2^3K/Q, which is defective in movement but not rhodopsin binding. Since the retinal degeneration in norpA is largely rescued in arr2^3K/Q flies, these results suggest that apoptosis in norpA results from endocytosis of Arr2/rhodopsin complexes rather than a defect in Arr2/rhodopsin binding. This conclusion is further supported by the finding that there was even greater suppression of the norpA degeneration in an arr2^3K/Q than in an arr2⁵ null background (Lee, 2003).

Molecular basis of amplification in Drosophila phototransduction: Roles for G Protein, Phospholipase C, and Diacylglycerol kinase

In Drosophila photoreceptors, the amplification responsible for generating quantum bumps in response to photoisomerization of single rhodopsin molecules has been thought to be mediated downstream of phospholipase C (PLC), since bump amplitudes were reportedly unaffected in mutants with greatly reduced levels of either G protein or PLC. It is found that quantum bumps in such mutants are reduced ~3- to 5-fold but are restored to near wild-type values by mutations in the rdgA gene encoding diacylglycerol kinase (DGK) and also by depleting intracellular ATP. The results demonstrate that amplification requires activation of multiple G protein and PLC molecules; they identify DGK as a key enzyme regulating amplification, and implicate diacylglycerol as a messenger of excitation in Drosophila phototransduction (Hardie, 2002).

Many photoreceptors generate discrete responses to effective absorptions of single photons, known as quantum bumps. In Drosophila, these represent simultaneous activation of about 15 light-sensitive channels generating an inward current of ~10 pA. The response is remarkable for its speed, with latencies as short as 20 ms and bump halfwidths of ~20 ms. These kinetics are about 100 times faster than in toad rods recorded at similar temperatures, about 10 times faster than mammalian rods recorded at 37°C, and in general, fly photoreceptors are considered to have the fastest known G protein-coupled signaling cascades. The events leading to quantum bump generation in Drosophila have been inferred from a variety of physiological, biochemical, and genetic evidence. Photoisomerized rhodopsin activates a heterotrimeric G protein (Gq class) releasing the alpha subunit, which in turn activates phospholipase C (PLCß4 isoform) encoded by the norpA gene. By a still unknown mechanism, activation of PLC leads to the opening of at least two classes of Ca²⁺ permeable channels, TRP and TRPL. These are the prototypical members of the TRP ion channel superfamily responsible for a wide variety of Ca²⁺ influx pathways throughout the body. Recent evidence suggests that the Drosophila channels, as well as some vertebrate TRP homologs, may be activated by lipid second messengers rather than by InsP₃. Candidates include diacylglycerol (DAG), polyunsaturated fatty acids (PUFAs), which are DAG metabolites, or a reduction in phosphatidyl inositol 4,5 bisphosphate (PIP₂). Following activation of the first channel(s), Ca²⁺ influx mediates rapid positive and negative feedback, which is required for both amplification and rapid termination of the quantum bump. Most, if not all, elements of the phototransduction cascade are located in the ~30,000 tightly packed microvilli, each only 60 nm in diameter and 1–2 µm long, which together form the light-guiding rhabdomere. Quantitative Western analysis suggests that there are about 25 TRP channels per microvillus, which corresponds closely to the number of channels activated during the quantum bump. It is therefore plausible that quantum bump generation is restricted to a single microvillus, representing activation of all or most of the available channels (Hardie, 2002).

In contrast to the situation in vertebrate rods, recent evidence has led to the proposal that all amplification in this system is mediated downstream of PLC. The main evidence for this view is that quantum bump amplitude is reported to be unaffected in hypomorphic mutants of G protein (Gaq) and PLC (norpA) where protein levels are reduced to levels (<1%) such that there may often be no more than a single G protein or PLC molecule in each microvillus. This led to the conclusion that levels of G protein do not actively contribute to the gain of the single photon response and that the G protein must act as a 'molecular switch,' triggering bump generation (Hardie, 2002).

In the present study, this view has been questioned because of the observation of spontaneous events in the dark in WT flies. Although the data suggested they were due to spontaneous activation of G proteins, they are much smaller than quantum bumps, seemingly inconsistent with single G proteins triggering full-sized bumps. Quantum bumps were therefore systematically reinvestigated in both Gaq and norpA hypomorphs; contrary to previous reports, bump amplitudes were much reduced, suggesting that there is substantial amplification upstream of PLC. The discrepancy with earlier studies is resolved by showing that bump amplitude in both Gaq and norpA mutants could be increased to near WT levels by omitting ATP from the whole-cell recording pipette. Finally, DAG kinase has been identified as the critical ATP-dependent factor, strongly supporting the proposal that DAG (or its downstream metabolites) is the excitatory messenger responsible for channel activation (Hardie, 2002).

The current view that amplification in the Drosophila phototransduction cascade is mediated downstream of PLC is based largely on reports that bump amplitude and timecourse are unaffected in mutants of G protein or PLC. However, after testing an extensive range of mutants of both G protein and PLC, it was found that in all cases quantum bump amplitude was greatly reduced. Although these results seemed to directly contradict earlier studies, it was found that when ATP was omitted from the electrode, bump amplitude approached WT values. This almost certainly accounts for the apparently conflicting results (Hardie, 2002).

Cook (2000) analyzed bump in detail only from norpA^C1094S flies, which this study has found to have among the largest bumps of the norpA alleles tested (~3 pA with occasional bumps as large as 10 pA). Since Cook (2000) excluded events less than 3 pA in amplitude from their analysis, their results are not necessarily in conflict with the results of this study: small bumps are indeed found, e.g., with norpA^P57 under similar recording conditions. Most significantly, the effect of removing ATP could be closely mimicked by mutations in the rdgA gene encoding DGK. These results indicate that amplification in Drosophila is critically dependent on activation of multiple G protein and PLC molecules and identify DGK as a key enzyme regulating bump amplitude and inactivation (Hardie, 2002).

It is now concluded that amplification in Drosophila phototransduction is critically dependent upon activation of multiple G proteins and PLC molecules. Assuming linear summation, the difference in bump current integral between WT and the most severe Gq and norpA hypomorphs suggests that at least five PLCs need be activated in order to generate a typical WT bump. Since quantum bumps in Drosophila correspond to the simultaneous opening of only about 15 channels at the peak of the bump, amplification at the level of the G protein may in fact represent the major component of amplification in Drosophila. Interestingly, a similar number of G proteins (about eight) are believed to be activated in Limulus ventral photoreceptors, although amplification downstream of PLC dominates in these cells -- probably by very distinct mechanisms (Hardie, 2002).

Significantly, lowering ATP did not further increase bump amplitude in WT photoreceptors, suggesting that the bump-generating machinery is saturated in dark-adapted photoreceptors. This can be understood if quantum bumps represent activation of all available channels within the microvillus. The suggestion that the unit of signaling underlying the quantum bump is the microvillus is also consistent with the finding that the number of channels activated during the quantum bump corresponds closely to the number predicted per microvillus from quantitative Western analysis (Hardie, 2002).

Is DAG the excitatory messenger? The essential role of PLC in Drosophila phototransduction is well established, but the downstream mechanisms responsible for gating the light-sensitive channels remain controversial. Accumulating evidence, including the lack of phenotype in mutants of the only InsP₃ receptor gene known in Drosophila, suggests that InsP₃ may not be involved in excitation. Additional consequences of PLC activation include generation of DAG and a reduction in PIP₂, both of which are also currently under discussion as excitatory messengers for some vertebrate TRP homologs. Both TRP and TRPL channels can be activated by polyunsaturated fatty acids (PUFAs), which might be released from DAG by a DAG lipase. It has been found that heterologously expressed TRPL channels can also be activated by DAG, raising the possibility that DAG may be the endogenous transmitter, with PUFAs mimicking their action. However, at least some of the actions of DAG and PUFAs may be indirect via activation of endogenous PLC; activity of TRPL channels in patches is suppressed by application of PIP₂, suggesting PIP₂ depletion as a potential contributory factor to channel gating (Hardie, 2002).

The analysis of the DGK mutant rdgA in this study provides strong independent evidence for an excitatory role for DAG. TRP and TRPL channels in rdgA mutants are constitutively active and degeneration is largely prevented in rdgA;trp double mutants. The response to light is also rescued in rdgA;trp, revealing a deactivation defect, suggesting a role for DGK in response termination, at least with respect to TRPL channels. These results would be consistent with a role for DAG in excitation; however, DGK is also the first enzyme in the PIP₂ recycling pathway, so that PIP₂ levels may also be affected in the rdgA mutant. Furthermore, the remaining TRPL channels were still constitutively active in the rdgA;trp double mutant, and light responses could only be compared to controls in pupae during a very narrow developmental time window (Hardie, 2002).

In the present study, rdgA double mutants were generated with both Galphaq¹and norpA, allowing analysis in adult flies with intact TRP and TRPL channel function. Strikingly, bump amplitudes in both norpA and Galphaq¹ are restored to WT levels by rdgA mutations, also showing defects in inactivation. In addition, quantum efficiency (Q.E. -- the percentage of absorbed photons eliciting a bump) is enhanced, resulting in some cases in massive (~100-fold) overall increases in sensitivity. Since the evidence indicates that these rdgA phenotypes are not due to reduced PIP₂ levels, this is interpreted as compelling evidence for the role of DAG as messenger of excitation. DAG levels are dynamically determined by the balance between PLC activity (generating DAG from PIP₂) and DGK activity (converting DAG to PA). It seems that when only one PLC molecule is activated, DAG is metabolized too quickly for threshold levels to be reached, except perhaps in the immediate vicinity of the activated PLC. However, if DGK is inactivated by the rdgA mutation or by depleting ATP, then DAG can reach threshold more readily and also diffuse to activate more distant channels so that quantum efficiency and quantum bump amplitudes approach WT values. (Note that the reduction in Q.E. in Gaq¹ has been attributed to most activated rhodopsins being inactivated before they have a chance to encounter a rare G protein; the results of this study suggest that a major factor in the reduction of Q.E. is that, despite activating a PLC, most single activated G proteins result in insufficient DAG generation to overcome bump threshold). Despite these arguments, a contributory role of PIP₂ to channel regulation cannot be excluded. For example, it is conceivable that channels are bound to PIP₂ in the closed state, that channel activation involves exchange of PIP₂ for DAG, and that channel closure following excitation may involve rebinding of PIP₂ (Hardie, 2002).

Multiple, sequential G protein activation is well established as a mechanism of amplification in vertebrate phototransduction. Although it has been concluded that multiple G protein activation is also required for amplification in Drosophila, the molecular strategies remain qualitatively and quantitatively distinct. In rods, each activated G protein rapidly encounters a PDE, which immediately begins to hydrolyze cGMP. The cGMP concentration is sensed continuously by the light-sensitive channels, which progressively close as upward of 100 G protein and PDE molecules are recruited by random diffusional encounters with rhodopsin. This gives rise to quantum bumps with very short latencies, but which rise gradually over a time course of ~1 s in toad or ~100 ms in mammalian rods. In Drosophila and other microvillar photoreceptors, there is a finite and variable latency (~20–100 ms in Drosophila) followed by an abrupt (~10 ms) rising phase, indicative of a threshold and positive feedback—features not found in vertebrate photoreceptors. At rest in the dark, the channels are closed, and the latency presumably represents the time taken for diffusional encounters of, perhaps, five to ten G proteins with rhodopsin and PLC and then for sufficient second messenger (DAG or its PUFA metabolites) to accumulate to activate the first channel. Because of the restricted volume of the microvillus, Ca²⁺ influx via the first channel raises Ca²⁺ rapidly throughout the microvillus. If other channels in the microvillus are already exposed to subthreshold concentrations of DAG generated by other PLC molecules, it is proposed that the raised Ca²⁺ sensitizes these channels (e.g., by increasing the affinity of the channel for DAG/PUFA), resulting in an explosive positive feedback which activates all or most of the channels in the microvillus over a time course of ~10 ms. Ca²⁺, which reaches concentrations of at least 200 µM, then mediates negative feedback terminating the bump. Finally, there is believed to be a refractory period lasting ~100 ms, while the Ca²⁺ is cleared by diffusion and/or Na⁺/Ca²⁺ exchange and necessary biochemical steps of inactivation (e.g., Rh-arrestin binding, GTPase activity, clearance of DAG, resynthesis of PIP₂) run their course (Hardie, 2002).

In summary, the results indicate that amplification in Drosophila phototransduction is critically dependent upon activation of multiple G proteins and PLC molecules, identify DGK as a key enzyme regulating amplification, and strongly support the identification of DAG as messenger of excitation in Drosophila phototransduction. PLC has long been recognized as the effector enzyme in invertebrate phototransduction, playing an analogous role to PDE in vertebrate rods (although PLC generates the active transmitter, while PDE degrades it). The results now suggest that DGK is the key enzyme controlling the supply of second messenger in Drosophila and thus plays an analogous role to guanylate cyclase. Ca²⁺-dependent regulation of guanylate cyclase is a key mechanism of light adaptation and response termination in vertebrate rods. It will be interesting to see whether similar regulation of DGK is involved in regulating sensitivity and kinetics during light adaptation in Drosophila (Hardie, 2002).

Seasonal behavior in Drosophila melanogaster requires the photoreceptors, the circadian clock, and phospholipase C

Drosophila locomotor activity responds to different seasonal conditions by thermosensitive regulation of splicing of a 3' intron in the period mRNA transcript. The control of locomotor patterns by this mechanism is primarily light-dependent at low temperatures. At warmer temperatures, when it is vitally important for the fly to avoid midday desiccation, more stringent regulation of splicing is observed, requiring the light input received through the visual system during the day and the circadian clock at night. During the course of this study, it was observed that a mutation in the no-receptor-potential-A(P41) [norpA(P41)] gene, which encodes phospholipase-C, generates an extremely high level of 3' splicing. This cannot be explained simply by the mutation's effect on the visual pathway and suggests that norpA(P41) is directly involved in thermosensitivity (Collins, 2004).

The proportion of per transcripts that were spliced at 18°C and 29°C, averaged over several LD 12:12 cycles was examined in Canton-S WT and per⁰¹, tim⁰¹, cry^b, and per⁰¹; cry^b mutant backgrounds. In all backgrounds splicing levels fall as the temperature rises, with 40%-60% of transcripts spliced at 18°C and 20%-45% at 29°C. However, not all genotypes react in the same way to temperature changes (Collins, 2004).

The smallest but nevertheless significant effect of temperature on splicing levels is observed in per⁰¹; cry^b, suggesting that the temperature-sensing system for splicing may be compromised in the double mutant. A significant temperature x time effect reveals that the temporal patterns of cycling differ among temperatures, and the absence of any other significant interactions suggests that all genotypes respond similarly. There is very little evidence for a significant day/night cycle in the proportion of per transcripts that are spliced at 18°C, but at 29°C, all genotypes reveal a higher level of splicing post lights off (ZT12) compared to the trough at ZT8. At 18°C, the per⁰¹ and tim⁰¹ mutations have no significant effect on the level of splicing of per mRNA compared to WT. However in cry^b flies, splicing levels are significantly elevated, particularly after lights off. This is also the case when per⁰¹; cry^b is compared to WT. At 29°C, splicing levels are generally 5%-10% higher in per⁰¹, tim⁰¹, and cry^b mutants compared to WT in the light, but 15%-20% higher after lights off at ZT12. This suggests that in the presence of light, splicing levels are reduced due largely to a clock-independent mechanism. In darkness, the clock and Cry become critical for maintaining this low splicing level at high temperatures (Collins, 2004).

The double mutant per⁰¹; cry^b shows a highly significant increase in splicing of ~20% throughout the day/night cycle compared to WT. Thus, at high temperature, either the presence of the circadian photoreceptor Cry or a functional circadian clock is sufficient to largely repress daytime splicing. With both eliminated, daytime splicing levels are elevated. In contrast, repression of splicing in the absence of light requires the circadian clock plus Cry. It seems somewhat counterintuitive that Cry, which is activated by light, plays a more prominent role in repressing splicing at night than it does during the day (Collins, 2004).

Cry is likely to be a dedicated circadian photoreceptor yet at 29°C, splicing is repressed during the light phase even in cry^b. This suggests that the light input to the splicing machinery cannot be primarily mediated by Cry. To confirm that light represses splicing, the effects that short photoperiods and constant darkness (DD) have on splicing levels in WT was investigated. There is a significant effect of reducing photoperiod on the splicing level with an elevated level of splicing in DD compared to LD 12:12, and similarly in LD 6:18, splicing levels are enhanced. Because the repression of splicing by light in LD 12:12 at 29°C does not require the presence of Cry, whether the visual system plays a role in setting the splicing level was investigated by examining the splicing of per transcripts in the mutants gl^60j and norpA^P41 (Collins, 2004).

The proportion of per mRNA transcripts that are spliced at both temperatures is increased in both the norpA^P41 and gl^60j backgrounds compared to WT. At 18°C, ~65% of per transcripts are spliced in norpA^P41 and ~60% in gl^60j, whereas at 29°C, these levels fall to ~55% and ~40%, respectively. Apart from a marginal difference between norpA^P41; cry^b, and norpA^P41 at 18°C, there are no significant effects for either norpA^P41 or gl^60j when combined with cry^b. These results indicate that the visual system rather than Cry is primarily responsible for the light-dependent repression of splicing. Unlike WT, per splicing levels do not rise after lights off at 29°C in either norpA^P41 or gl^60j (Collins, 2004).

Interestingly, in gl^60j, there is a 20% difference between the splicing levels at different temperatures (60%-40%), whereas in norpA^P41, this difference is reduced to 10% (65%-55%). The difference in gl^60j is similar to that seen in WT (45%-25%). Thus per splicing in norpA^P41 is relatively insensitive to temperature changes. It is also clear that the level of splicing in norpA^P41 is significantly higher at all times and temperatures than gl^60j. Therefore, the effect of norpA^P41 on splicing is greater than that of gl^60j_, despite gl^60j being the more severe visual mutant (Collins, 2004).

Locomotor activity profiles of all genotypes were also monitored at 18°C and 29°C. Because each genotype shows a higher level of splicing at 18°C than at 29°C, it would be predicted that this would generate an earlier evening activity peak at 18°C. This is the case for WT, cry^b and norpA^P41, but not for norpA^P41; cry^b or gl^60j, where despite elevated splicing levels at higher temperatures, there is no difference in the phase of activity. gl^60j cry^b does not entrain to LD cycles at 25°C, so was not included in this analysis (Collins, 2004).

The average proportion of per transcripts that are spliced at 18°C rises from WT (45%) to cry^b (50%) to gl^60j (60%) to norpA^P41 (65%), and at 29°C from WT (25%) to gl^60j and cry^b (~35%) to norpA^P41 (55%). If the per splicing level is the only determinant of evening locomotor peak position, then a similar progression in the timing of this peak would be expected. The evening activity peaks of these different genotypes at 18°C and 29°C were compared. For norpA^P41, cry^b, and WT, there is an inverse relationship between average splicing levels and the position of the activity peak at 18°C, with norpA^P41 and cry^b having similarly advanced activity peaks compared to WT. At 29°C, the same inverse relationship holds, with norpA^P41 advanced compared to cry^b, which is in turn earlier than WT. Thus, those genotypes that show temperature-dependent changes in their evening activity generally display a correlation between average per splicing levels and the timing of the evening activity peak of the following day. Conversely, norpA^P41; cry^b and gl^60j,, which show no significant differences in the phase of evening activity at different temperatures, have high splicing levels but relatively delayed evening activity peaks (Collins, 2004).

These observations raise the question of why the splicing level does not always relate to the timing of the evening locomotor activity peak, as in gl^60j and norpA^P41; cry^b. Thus the per RNA profiles of gl^60j and norpA^P41 were compared to WT. Because per does not cycle in cry^b whole head homogenates, the underlying cycle in this background was not examined. WT and norpA^P41 show similar profiles, with an earlier per mRNA peak and higher overall level of per at the lower temperature. In contrast, there is no cycle in gl^60j at either temperature, and levels of per are significantly different from WT and norpA^P41 (Collins, 2004).

Therefore, to entrain locomotor behavior to different seasons, the fly's clock must respond to changes in both light and temperature. This is mediated through a molecular switch, whereby increases in temperature repress the splicing of an intron within the 3' UTR of per, delaying the onset of evening locomotor activity. Light also represses splicing, with higher splicing levels seen in shorter photoperiods, allowing locomotor activity to be fine-tuned to any given set of photoperiodic and temperature conditions. During the first day of DD, the level of splicing rises continuously. This is presumably because at the beginning of DD, the level of splicing is set low from the previous day's light input. Normally the light from the next day maintains this repressed level of splicing, but because this light input is absent, the repression of splicing is lifted, leading to a gradual rise in splicing levels (Collins, 2004).

The most obvious source for light input into the splicing machinery is the circadian photoreceptor Cry. However, analysis of the splicing levels in cry^b shows that, although this mutation has an effect on splicing levels at 18°C, this effect is marginal and is seen only after lights off. This implies (1) that any function of Cry in the repression of splicing is not via the activation of this molecule by light; (2) because Cry is relatively dispensable for circadian locomotor rhythmicity per se, it also suggests that any minor role in splicing at low temperature is unrelated to the functioning of the clock. As the temperature rises, Per, Tim, and Cry all become involved in the regulation of per mRNA splicing. At 29°C, all three mutants show the same splicing phenotype, with ~30% of transcripts spliced during the day, but at night splicing is enhanced to ~45%. Although Per, Tim, and Cry are known to associate in light conditions, Cry and Tim can also associate in darkness, so it is not unexpected that the elimination of any one of the three proteins has a similar effect. Night time is also when the levels of these proteins are at their highest, and therefore any effects would be maximal (Collins, 2004).

At 29°C and in the presence of light, the levels of splicing in per⁰¹; cry^b are elevated above those of either single mutant, which are themselves similar to WT. This suggests that the presence of either Per or Cry is required for light to repress splicing at 29°C. After lights off, the elevated levels of splicing of per are very similar in per⁰¹, tim⁰¹, cry^b, and per⁰¹; cry^b. Therefore Per, Tim, and Cry probably work together to repress splicing in the dark at 29°C. An alternative view for the virtually identical per⁰¹, tim⁰¹, and cry^b splicing levels at 29°C is that this reflects a masking effect of light, so that exogenous LD cycles have a greater effect on splicing at night compared to WT, which shows a modest but significant day-night rhythm. Such stronger masking effects on locomotor behavior have also been observed in cry^b mutants, but any mechanism that might relate or explain these observations remains obscure (Collins, 2004).

The examination of whole head homogenates means that the majority of biological material is derived from the eyes so may not represent exactly what occurs in the pacemaker neurons. The eyes are peripheral clocks, and the cry^b mutation stops the cycling of the clock in whole head homogenates, although cycling continues in the pacemaker cells. One possibility is that the splicing observed in cry^b does not truly reflect the role of Cry in setting splicing levels but is instead a consequence of the clock having stopped in the eyes, thus explaining why per⁰¹, tim⁰¹, and cry^b all show the same splicing phenotype. However, if this splicing phenotype is simply what happens when the clock stops, then per⁰¹; cry^b should show the same splicing phenotype as either single mutant. This is not the case, because the daytime splicing in per⁰¹; cry^b at 29°C is dramatically elevated compared to either single mutant. Thus the splicing phenotypes of per⁰¹, tim⁰¹, and cry^b cannot simply be a result of the clock having stopped. This means that it is the presence of these proteins, rather than their clock-dependent cycling, that is important to the regulation of per splicing levels (Collins, 2004).

In gl^60j, there is no per mRNA cycle in whole head homogenates. This means that in the majority of cells in the gl^60j head, the clock has either stopped or cells have become desynchronized. If the former is true, then splicing levels of gl^60j should resemble those of per⁰¹ or tim⁰¹, and this is clearly not the case. If the latter is true, this could prevent the observation of any splicing rhythm, but the level of splicing observed should still represent the average level of splicing in this mutant background, which is clearly significantly different from WT. In any case, splicing levels observed in all visual mutants are likely to represent the effect of removing visual photoreception, because these elevated levels are similar to those observed in WT in DD (Collins, 2004).

norpA^P41 and gl^60j have considerably higher splicing levels than WT and cry^b mutants at both temperatures, indicating that information received via the visual system rather than Cry drives this repression of splicing, which is borne out by analysis of gl^60j cry^b and norpA^P41; cry^b double mutants. The splicing levels of gl^60j and gl^60j cry^b are similar at both temperatures, which is also true of norpA and norpA^P41; cry^b at 29°C. At 18°C, there is slightly more spliced per RNA in norpA^P41; cry^b than in the norpA^P41 single mutant, reflecting the earlier result where cry^b showed a marginal enhancement of splicing at cooler temperatures. These results also demonstrate that unspecific genetic background effects are not responsible for this marginal effect of cry^b, because the double mutant background should make any interacting loci heterozygous. This lack of significant background effects in determining overall splicing levels has been confirmed by examining several natural European D. melanogaster lines. All mutants studied here show the same significantly enhanced splicing patterns when compared to any of the wild-caught isolates (Collins, 2004).

Unlike the clock and cry^b mutants, there is no day-night difference in splicing levels at 29°C in either gl^60j or norpA^P41. One possibility is that visual system structures are required for the repression of splicing even in the dark, hence the overall elevated splicing levels in norpA^P41 and gl^60j at all times. This would be surprising, because such a role would obviously have to be light independent. More likely, the light input received through the eyes sets the splicing level during the day, and the clock maintains this repression at night. Thus, if the visual input is removed or reduced, as in DD, gl^60j, or norpA^P41 mutants, or in shorter photoperiods, then the subsequent splicing level is set higher. The difference in roles between cry and the visual system on per splicing levels may also partly explain recent observations that cry^b mutants are able to adapt the timing of locomotor activity to long and short photoperiods, whereas flies with defective visual photoreception, including gl^60j, are not (Collins, 2004).

Interestingly, although gl^60j is the more severe visual mutant, norpA^P41 has significantly higher per splicing levels than gl^60j at both 18°C and 29°C. Additionally, whereas the difference between splicing levels at 18°C and 29°C is maintained in gl mutants (~65% and ~45% of transcripts spliced vs. ~45% and 25% in WT at 18°C and 29°C, respectively), this is greatly reduced in norpA^P41 (65% and 55%). One possible explanation for this is that norpA may be a signaling molecule in the temperature-sensing pathway for the clock. The patterns of locomotor activity support a role for norpA in temperature sensing, with the norpA^P41 fly's locomotor patterns seemingly more sensitive to high temperatures than WT. Additionally, norpA^P41 evening locomotor activity peaks early at both 18°C and 29°C, and per mRNA splicing shows a corresponding elevation compared to WT. These are responses associated with low temperatures in WT D. melanogaster, and therefore norpA^P41 mutants behave as if they have an impaired ability to detect high temperatures. norpA^P41 flies still detect temperature changes (witness the altered evening peaks and splicing levels); they just react as if the temperature is colder than it actually is (Collins, 2004).

Thus, the enhanced per splicing seen in norpA^P41 may reflect a direct link between norpA-encoded PLC signaling and the temperature sensitivity of the splicing mechanism, independent of norpA visual function. In the phototransduction cascade, rhodopsin activates a G-protein isoform that in turn activates the PLC encoded by norpA. As a result of this activation, Ca²⁺ permeable light-sensitive channels are opened, including members of the transient receptor potential (TRP) class. Recently it has been demonstrated that dANKTM1, a D. melanogaster TRP channel, is activated by temperatures from 24°C to 29°C. In addition, D. melanogaster painless mutant larvae have a disrupted TRP channel and display defective responses to thermal stimuli. Because several TRP family members act as thermal sensors in mammals, TRP channels appear to have an ancient heat-sensing function that is retained in both vertebrates and invertebrates. Given that this study has identified a heat-sensing role for norpA, and norpA is known to activate TRP channels in photoreception, it is not unreasonable to suppose that norpA plays a general role in responses to temperature stimuli (Collins, 2004).

per splicing levels may also impact on aspects of behavior other than the timing of evening locomotor activity. For instance, the free-running period of norpA^P41 is ~1 h shorter than WT. The splicing levels of per mRNA are greatly elevated in this background, and elevated splicing is predicted to advance the Per protein cycle and thus speed up the clock. In fact, the splicing mechanism should have the effect of speeding up the clock at colder temperatures and slowing it down at high temperatures, thereby providing a potential basis for temperature compensation (Collins, 2004).

The position of the evening activity peak at different temperatures moves in different mutant backgrounds. For WT, norpA^P41, and cry^b, the level of splicing appears to correlate with the position of the evening activity peak at different temperatures. At 18°C, there is a small but significantly greater relative amount of spliced per RNA in cry^b than in WT, resulting in the earlier evening activity peak seen in cry^b flies. This difference in per splicing is greatest after lights off at both temperatures. This is when Per levels will be rising, because Tim is present for Per stabilization, so enhancement of Per accumulation by elevated per splicing is likely to have its most noticeable effect around dusk or early evening. A similarly consistent situation is seen in norpA^P41: there is more spliced per mRNA present at 18°C (65%) than 29°C (55%), accounting for the earlier peak of evening activity at 18°C. Additionally these levels are higher than those seen in either WT (45% and 25% per transcripts spliced at each temperature) or cry^b (55% and 40%) and relates to the earlier phases of locomotor activity seen in norpA^P41 compared to the other genotypes. However, at 18°C there is more spliced per in norpA^P41 than in cry^b, but the evening activity peak occurs at the same time. The simplest explanation is that there is a limit to how early the evening activity peak can occur, no matter what the per splicing level, because splicing alters the accumulation of Per protein; this is limited by the light-dependent degradation of Tim. Therefore, in general, the level of splicing determines when the peak level of locomotor activity will occur (Collins, 2004).

The level of splicing of the per intron cannot be the only determinant of evening peak position, because the relationship between the per splicing level and evening activity peak position breaks down in norpA^P41; cry^b and gl^60j, where there are different levels of splicing at the two temperatures but no corresponding difference in the evening peak position. When the underlying per mRNA cycles of gl^60j, norpA^P41, and WT flies were analyzed at 18°C and 29°C, it was found that whereas per levels cycle in norpA^p41 and WT, this cycle is lost in gl^60j. If there is no underlying per RNA cycle, then there is no mRNA peak to be advanced or delayed by splicing (Collins, 2004).

At the cellular level, although gl is not a clock component, when mutated, it eliminates a number of clock-expressing cells within the head, including the eyes, ocelli, Hofbauer-Buchner (H-B) eyelet, and the dorsal neuron 1 (DN1) cells. Despite this, the primary effect on the clock is to remove most of the visual entrainment pathway, but the clock in the key pacemaker cells of gl^60j mutants must still be functional, because behavior still entrains to LD cycles and remains rhythmic in DD. It is significant that the crosstalk between different classes of clock cells is essential for the generation of robust behavioral rhythms. Thus loss of the overall per mRNA rhythm may be a consequence of disrupting this network in gl^60j, and, while leaving the basic system intact, this affects the more subtle temperature-sensitive aspects of entrainment. A similar argument based on an interruption of the entrainment network can also be proposed to explain the corresponding results with norpA^P41; cry^b double mutants, because in this case per mRNA is assumed to be noncycling because of the cry^b background. However, the locomotor behavior of cry^b single mutants remains thermosensitive even though overall per mRNA is noncycling. Thus, only when the photoreceptive pathway and mRNA cycle are both compromised (as in gl and norpA^P41; cry^b) is locomotor behavior insensitive to temperature-dependent changes in per splicing levels (Collins, 2004).

A model is presented of how light and temperature may set the splicing level of the clock. How temperature is detected by the splicing machinery is not yet clear, but there is compelling evidence that norpA plays a role. At low temperatures, the splicing level is primarily set by light via the visual system rather than Cry, which is then remembered during the night. In longer periods of darkness such as in DD, this memory decays, and splicing levels begin to rise. Thus the visual system represses splicing by enhancing the effects of an unknown repressor molecule(s) that is sensitive to temperature change and the norpA PLC. At high temperatures, the regulation of splicing is more stringent and complex and recruits the circadian clock. Again, the light input received through the visual system sets the low splicing level during the day. This appears to also depend on the presence of at least two of the three molecules, Per, Tim, or Cry, because elimination of any one of these gives a barely detectable daytime rise in splicing, reflecting the very low levels of Per, Tim, and Cry at this time. However, elimination of both Per and Cry in the per⁰¹; cry^b double mutant lifts all light-dependent repression during the day (Collins, 2004).

At night, the level of splicing set during the day by the visual system is again remembered and maintained by the clock at night. If per, tim, or cry is eliminated, then this repression of splicing is lost at night, generating the day/night difference in splicing levels. In gl^60j cry^b or norpA^P41; cry^b, because there is no visual light input during the day, there is no splicing level for the clock to remember, and therefore there is no day/night difference in splicing levels. Thus at high temperature, the visual system activates the repressor molecule during the day, and the clock maintains this activation at night. It is assumed that recruiting the clock at high temperature to inhibit per splicing is required to ensure that the fly's locomotor/foraging behavior is adaptive and does not encroach on those times of the day when there would be a significant risk of desiccation (Collins, 2004).

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D

The Interactive Fly resides on the
Society for Developmental Biology's Web server.