Immunoreactivities to three circadian clock proteins in two ground crickets suggest interspecific diversity of the circadian clock structure

The closely related crickets Dianemobius nigrofasciatus and Allonemobius allardi exhibit similar circadian rhythms and photoperiodic responses, suggesting that they possess similar circadian and seasonal clocks. To verify this assumption, antisera to Period (PER), Doubletime (DBT), and Cryptochrome (CRY) were used to visualize circadian clock neurons in the cephalic ganglia. Immunoreactivities referred to as PER-ir, DBT-ir, and CRY-ir were distributed mainly in the optic lobes (OL), pars intercerebralis (PI), dorsolateral protocerebrum, and the subesophageal ganglion (SOG). A system of immunoreactive cells in the OL dominates in D. nigrofasciatus, while immunoreactivities in the PI and SOG prevail in A. allardi. Each OL of D. nigrofasciatus contains 3 groups of cells that coexpress PER-ir and DBT-ir and send processes over the frontal medulla face to the inner lamina surface, suggesting functional linkage to the compound eye. Only 2 pairs of PER-ir cells (no DBT-ir) were found in the OL of A. allardi. Several groups of PER-ir cells occur in the brain of both species. The PI also contains DBT-ir and CRY-ir cells, but in A. allardi, most of the DBT-ir is confined to the SOG. Most immunoreactive cells in the PI and in the dorsolateral brain send their fibers to the contralateral corpora cardiaca and corpora allata. The proximity and, in some cases, proven identity of the PER-ir, DBT-ir, and CRY-ir perikarya are consistent with presumed interactions between the examined clock components. The antigens were always found in the cytoplasm, and no diurnal oscillations in their amounts were detected. The photoperiod, which controls embryonic diapause, the rate of larval development, and the wing length of crickets, had no discernible effect on either distribution or the intensity of the immunostaining.     

Neurons projecting to the retrocerebral complex of the adult blow fly, Protophormia terraenovae

Anatomical study of neurons projecting to the retrocerebral complex of the adult blow fly, Protophormia terraenovae, was done by NiCl2 filling and immunocytochemistry. Retrograde filling through the cardiac-recurrent nerve labeled three groups of neurons in the brain/subesophageal ganglion: (1) paramedial clusters of the pars intercerebralis, (2) neurons in each pars lateralis, and (3) neurons in the subesophageal ganglion. The pars intercerebralis neurons send prominent axons into the median bundle and exit from the brain via the contralateral nervus corporis cardiaci. Based on the projection pattern, two types of the pars lateralis neurons can be distinguished: the most lateral pairs of neurons contralaterally extend through the posterior lateral tract and the remainder ipsilaterally extend through the posterior lateral tract. The neurons in the subesophageal ganglion run through the contralateral nervus corporis cardiaci. The dendritic arborization of the pars intercerebralis and pars lateralis neurons is restricted to the superior protocerebral neuropil and to the anterior neuropil of the subesophageal ganglion where the neurons in the subesophageal ganglion also project. Retrograde filling from the corpus allatum indicated that the pars lateralis neurons and a few pars intercerebralis neurons project to the corpus allatum, but that the neurons in the subesophageal ganglion do not. Orthograde filling from the pars intercerebralis and staining by paraldehyde-thionin/paraldehyde-fuchsin indicated that the pars intercerebralis neurons project primarily to the corpus cardiacum/hypocerebral ganglion complex. Immunostaining with a polyclonal antiserum against diapause hormone, a member of the FXPRLamide family, suggests that some of the subesophageal ganglion neurons contain FXPRLamide-like peptides.     

Molecular characterization and distribution of CYCLE protein from Athalia rosae

cDNA encoding CYCLE (CYC) from the coleseed sawfly, Athalia rosae (Hymenoptera, Symphyta), was amplified by PCR. This is a first determination of hymenopteran CYC structure. ArCYC had an overall identity of 66% with CYC of Anopheles gambiae and ca. 60% of Drosophila melanogaster. Structural investigation revealed that ArCYC contained characteristic motifs of: bHLH, PAS A, PAS B, PAC and BCTR. Detailed analysis indicated high conservation of these regions among insects. Northern blot analysis showed that the mRNA of ca. 3 kb was transcribed both in the head and in the rest of the body. Southern blot analysis suggested the presence of a single copy of the gene in the genome. Western blot indicates that the quantity of CYC protein does not fluctuate under LD 12:12 in either the head or the rest of the body. Immunocytochemical examination revealed CYC-like antigen in the pars intercerebralis, dorsolateral protocerebrum, dorsal optic tract, tritocerebrum of the brain and the subesophageal ganglion.     

Immunocytochemical studies on the central nervous system of the earthworm, Lumbricus terrestris

There are numerous aldehyde fuchsin (AF)-positive, neurosecretory cells of medium size (A cells) and a small number of large, AF-negative neurons (B cells) in the cortical layer of the cerebral ganglion. In the subesophageal ganglion, symmetrical groups of AF-positive cells lie ventrally. The peroxidase--antiperoxidase (PAP) method was used for the immunocytochemical study of substance P and ACTH in these ganglia. In addition, the presence of L-enkephalin and alpha endorphin could be confirmed. Using rabbit antibodies to substance P we found small immunoreactive neurons among negative A and B cells in the cerebral ganglion. The processes of these immunoreactive cells could be traced to the subcortical synaptic neuropil. With antibodies to ACTH, activity was visible in perikarya similar in size to A neurons. A part of the nerve terminals of the synaptic zone, some of the B neurons and further several nerve cells of the subesophageal ganglion reacted positively. Successive demonstration of substance P and ACTH on the same section showed that the two materials occurred in different cell types. Using antiopsin antibody in an indirect immunocytochemical test we observed strong reaction in numerous medium-sized perikarya and in nerve fibres of the synaptic zone of the cerebral ganglion, further in some neurons of the subesophageal and abdominal ganglia. In contrast to this result, the photoreceptor cells of the prostomium and cerebral ganglion were negative. Presumably, substance P is present in a perikaryon type hitherto unrecognized while ACTH and antiopsin reactions seem to be located first of all in A cells.     

Heterogeneous Expression of the Core Circadian Clock Proteins among Neuronal Cell Types in Mouse Retina

Circadian rhythms in metabolism, physiology, and behavior originate from cell-autonomous circadian clocks located in many organs and structures throughout the body and that share a common molecular mechanism based on the clock genes and their protein products. In the mammalian neural retina, despite evidence supporting the presence of several circadian clocks regulating many facets of retinal physiology and function, the exact cellular location and genetic signature of the retinal clock cells remain largely unknown. Here we examined the expression of the core circadian clock proteins CLOCK, BMAL1, NPAS2, PERIOD 1(PER1), PERIOD 2 (PER2), and CRYPTOCHROME2 (CRY2) in identified neurons of the mouse retina during daily and circadian cycles. We found concurrent clock protein expression in most retinal neurons, including cone photoreceptors, dopaminergic amacrine cells, and melanopsin-expressing intrinsically photosensitive ganglion cells. Remarkably, diurnal and circadian rhythms of expression of all clock proteins were observed in the cones whereas only CRY2 expression was found to be rhythmic in the dopaminergic amacrine cells. Only a low level of expression of the clock proteins was detected in the rods at any time of the daily or circadian cycle. Our observations provide evidence that cones and not rods are cell-autonomous circadian clocks and reveal an important disparity in the expression of the core clock components among neuronal cell types. We propose that the overall temporal architecture of the mammalian retina does not result from the synchronous activity of pervasive identical clocks but rather reflects the cellular and regional heterogeneity in clock function within retinal tissue.     

Mapping the cellular network of the circadian clock in two cockroach species

The German cockroach, Blattella germanica, and the double-striped cockroach, B. bisignata, are sibling species with a similar period sequence but a distinctive circadian rhythm in locomotion. The cell distribution of immunoreactivity (ir) against three clock-related proteins, Period (PER), Pigment Dispersing Factor (PDF), and Corazonin (CRZ), was compared between the species. The PER-ir cells tend to form clusters and are sprayed out in the central nervous system. Three major PER-ir cells are located in the optic lobes, which are the sites of the major circadian clock. They are interconnected with PER-ir axon bundles. Interestingly, the potential output signal of the circadian clock, PDF, is co-localized with PER in all three groups of cells. However, only two CRZ-ir cells and their axons are found in the optic lobes and they are not co-localized with PER-ir or PDF-ir cells and axons. Since only one circadian rhythm is expressed in locomotion, the time signals from both major clocks in optic lobes are coupled by connection with PDF-ir axons. A group of 3-4 PER-ir cells in the protocerebrum display typical characteristics of neurosecretary cells. In addition, there are numerous, small PER-ir and PDF-ir co-localized cells in the pars intercerebralis (PI), which have direct connections with the neurohemoorgan, corpora cardiaca, through PER-ir and PDF-ir axons. Based on these findings, the cellular connection shows a circadian control through the endocrine route. For the rest of central nervous system, only a few PER-ir and PDF-ir cells or axons are detected. This finding implies the circadian clock for locomotion is not located in subesophageal ganglion, thoracic or abdominal ganglia, but may use other neural messengers to pass on circadian signals. Since the overall distribution pattern of the clock cells are the same for B. germanica and B. bisignata, the possible explanation for the different expressions of locomotion between the species depends on genes downstream of per, pdf, and crz.     

Ectopic CRYPTOCHROME renders TIM light sensitive in the Drosophila ovary

The period (per) and timeless (tim) genes play a central role in the Drosophila circadian clock mechanism. PERIOD (PER) and TIMELESS (TIM) proteins periodically accumulate in the nuclei of pace-making cells in the fly brain and many cells in peripheral organs. In contrast, TIM and PER in the ovarian follicle cells remain cytoplasmic and do not show daily oscillations in their levels. Moreover, TIM is not light sensitive in the ovary, while it is highly sensitive to this input in circadian tissues. The mechanism underlying this intriguing difference is addressed here. It is demonstrated that the circadian photoreceptor CRYPTOCHROME (CRY) is not expressed in ovarian tissues. Remarkably, ectopic cry expression in the ovary is sufficient to cause degradation of TIM after exposure to light. In addition, PER levels are reduced in response to light when CRY is present, as observed in circadian cells. Hence, CRY is the key component of the light input pathway missing in the ovary. However, the factors regulating PER and TIM levels downstream of light/cry action appear to be present in this non-circadian organ.     

Hofbauer-Buchner eyelet affects circadian photosensitivity and coordinates TIM and PER expression in Drosophila clock neurons

Extraretinal photoreception is a common input route for light resetting signals into the circadian clock of animals. In Drosophila melanogaster, substantial circadian light inputs are mediated via the blue light photoreceptor CRYPTOCHROME (CRY) expressed in clock neurons within the brain. The current model predicts that, upon light activation, CRY interacts with the clock proteins TIMELESS (TIM) and PERIOD (PER), thereby inducing their degradation, which in turn leads to a resetting of the molecular oscillations within the circadian clock. Here the authors investigate the function of another putative extraretinal circadian photoreceptor, the Hofbauer-Buchner eyelet (H-B eyelet), located between the retina and the medulla in the fly optic lobes. Blocking synaptic transmission between the H-B eyelet and its potential target cells, the ventral circadian pacemaker neurons, impaired the flies' ability to resynchronize their behavior under jet-lag conditions in the context of nonfunctional retinal photoreception and a mutation in the CRY-encoding gene. The same manipulation also affected synchronized expression of the clock proteins TIM and PER in different subsets of the clock neurons. This shows that synaptic communication between the H-B eyelet and clock neurons contributes to synchronization of molecular and behavioral rhythms and confirms that the H-B eyelet functions as a circadian photoreceptor. Blockage of synaptic transmission from the H-B eyelet in the presence of functional compound eyes and the absence of CRY also results in increased numbers of flies that are unable to synchronize to extreme photoperiods, supplying independent proof for the role of the H-B eyelet as a circadian photoreceptor.     

Kinetics of doubletime kinase-dependent degradation of the Drosophila period protein

Robust circadian oscillations of the proteins PERIOD (PER) and TIMELESS (TIM) are hallmarks of a functional clock in the fruit fly Drosophila melanogaster. Early morning phosphorylation of PER by the kinase Doubletime (DBT) and subsequent PER turnover is an essential step in the functioning of the Drosophila circadian clock. Here using time-lapse fluorescence microscopy we study PER stability in the presence of DBT and its short, long, arrhythmic, and inactive mutants in S2 cells. We observe robust PER degradation in a DBT allele-specific manner. With the exception of doubletime-short (DBT(S)), all mutants produce differential PER degradation profiles that show direct correspondence with their respective Drosophila behavioral phenotypes. The kinetics of PER degradation with DBT(S) in cell culture resembles that with wild-type DBT and posits that, in flies DBT(S) likely does not modulate the clock by simply affecting PER degradation kinetics. For all the other tested DBT alleles, the study provides a simple model in which the changes in Drosophila behavioral rhythms can be explained solely by changes in the rate of PER degradation.     

Phosphorylation of period is influenced by cycling physical associations of double-time, period, and timeless in the Drosophila clock.

The clock gene double-time (dbt) encodes an ortholog of casein kinase Iepsilon that promotes phosphorylation and turnover of the PERIOD protein. Whereas the period (per), timeless (tim), and dClock (dClk) genes of Drosophila each contribute cycling mRNA and protein to a circadian clock, dbt RNA and DBT protein are constitutively expressed. Robust circadian changes in DBT subcellular localization are nevertheless observed in clock-containing cells of the fly head. These localization rhythms accompany formation of protein complexes that include PER, TIM, and DBT, and reflect periodic redistribution between the nucleus and the cytoplasm. Nuclear phosphorylation of PER is strongly enhanced when TIM is removed from PER/TIM/DBT complexes. The varying associations of PER, DBT and TIM appear to determine the onset and duration of nuclear PER function within the Drosophila clock.     

Light-dependent interaction between Drosophila CRY and the clock protein PER mediated by the carboxy terminus of CRY.

BACKGROUND: The biological clock synchronizes the organism with the environment, responding to changes in light and temperature. Drosophila CRYPTOCHROME (CRY), a putative circadian photoreceptor, has previously been reported to interact with the clock protein TIMELESS (TIM) in a light-dependent manner. Although TIM dimerizes with PERIOD (PER), no association between CRY and PER has previously been revealed, and aspects of the light dependence of the TIM/CRY interaction are still unclear. RESULTS: Behavioral analysis of double mutants of per and cry suggested a genetic interaction between the two loci. To investigate whether this was reflected in a physical interaction, we employed a yeast-two-hybrid system that revealed a dimerization between PER and CRY. This was further supported by a coimmunoprecipitation assay in tissue culture cells. We also show that the light-dependent nuclear interactions of PER and TIM with CRY require the C terminus of CRY and may involve a trans-acting repressor. CONCLUSIONS: This study shows that, as in mammals, Drosophila CRY interacts with PER, and, as in plants, the C terminus of CRY is involved in mediating light responses. A model for the light dependence of CRY is discussed.     

Isolation of twelve monoclonal antibodies against Ia and H-2 antigens. Serological characterization and reactivity with B and T lymphocytes

The cell hybridization technique was used for the production of 12 monoclonal antibodies against H-2K^k, H-2D^b, I-A^k and I-E^k antigens. The strain distribution pattern indicated that three antibodies reacted with new H-2 and Ia determinants, respectively, while the majority of determinants defined by the monoclonal antibodies showed good correlation with H-2 and Ia determinants described by conventional alloantisera.Monoclonal Ia antibodies showed strong reactivity with about 90% of surface IgM positive B cells, but not with T cells. In double fluorescence studies, both I-A and I-E determinants were always found to be coexpressed on the same B cells. When the high sensitivity of the fluorescence activated cell sorter was utilized, about 30 to 40% of purified lymph node T cells were found to carry both I-A and I-E antigens, although in a much lower density than B cells. In conclusion, monoclonal Ia antibodies appear to display the same serological and cellular reactivity pattern as do conventional antisera.