Development of the Molecular Circadian Clock and Its Light Sensitivity in Drosophila Melanogaster

2019 ◽  
Vol 34 (3) ◽  
pp. 272-282 ◽  
Author(s):  
Jia Zhao ◽  
Guy Robert Warman ◽  
Ralf Stanewsky ◽  
James Frederick Cheeseman
Keyword(s):  
Circadian Clock ◽  
Pupal Stage ◽  
Light Sensitivity ◽  
Embryonic Stage ◽  
Constant Darkness ◽  
Behavioral Experiments ◽  
Peripheral Tissues ◽  
Rhythmic Expression ◽  
Central Clock

The importance of the circadian clock for the control of behavior and physiology is well established but how and when it develops is not fully understood. Here the initial expression pattern of the key clock gene period was recorded in Drosophila from embryos in vivo, using transgenic luciferase reporters. PERIOD expression in the presumptive central-clock dorsal neurons started to oscillate in the embryo in constant darkness. In behavioral experiments, a single 12-h light pulse given during the embryonic stage synchronized adult activity rhythms, implying the early development of entrainment mechanisms. These findings suggest that the central clock is functional already during embryogenesis. In contrast to central brain expression, PERIOD in the peripheral cells or their precursors increased during the embryonic stage and peaked during the pupal stage without showing circadian oscillations. Its rhythmic expression only initiated in the adult. We conclude that cyclic expression of PERIOD in the central-clock neurons starts in the embryo, presumably in the dorsal neurons or their precursors. It is not until shortly after eclosion when cyclic and synchronized expression of PERIOD in peripheral tissues commences throughout the animal.

scholarly journals Period 2: A Regulator of Multiple Tissue-Specific Circadian Functions

2021 ◽  
Vol 14 ◽  
Author(s):  
Gennaro Ruggiero ◽  
Zohar Ben-Moshe Livne ◽  
Yair Wexler ◽  
Nathalie Geyer ◽  
Daniela Vallone ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Clock Genes ◽  
Central Element ◽  
Light Sensitivity ◽  
Loss Of Function ◽  
Tissue Specific ◽  
Rhythmic Expression ◽  
Period 2 ◽  
Circadian Clock Genes

The zebrafish represents a powerful model for exploring how light regulates the circadian clock due to the direct light sensitivity of its peripheral clocks, a property that is retained even in organ cultures as well as zebrafish-derived cell lines. Light-inducible expression of the per2 clock gene has been predicted to play a vital function in relaying light information to the core circadian clock mechanism in many organisms, including zebrafish. To directly test the contribution of per2 to circadian clock function in zebrafish, we have generated a loss-of-function per2 gene mutation. Our results reveal a tissue-specific role for the per2 gene in maintaining rhythmic expression of circadian clock genes, as well as clock-controlled genes, and an impact on the rhythmic behavior of intact zebrafish larvae. Furthermore, we demonstrate that disruption of the per2 gene impacts on the circadian regulation of the cell cycle in vivo. Based on these results, we hypothesize that in addition to serving as a central element of the light input pathway to the circadian clock, per2 acts as circadian regulator of tissue-specific physiological functions in zebrafish.

Zebrafish arylalkylamine-N-acetyltransferase genes – targets for regulation of the circadian clock

2006 ◽  
Vol 36 (2) ◽  
pp. 337-347 ◽  
Author(s):  
L Appelbaum ◽  
D Vallone ◽  
A Anzulovich ◽  
L Ziv ◽  
M Tom ◽  
...  
Keyword(s):  
Circadian Rhythm ◽  
Pineal Gland ◽  
Circadian Clock ◽  
Fluorescent Protein ◽  
Transgenic Fish ◽  
Constant Darkness ◽  
Rhythmic Expression ◽  
E Box

Daily rhythms of melatonin production are controlled by changes in the activity of arylalkylamine-N-acetyltransferase (AANAT). Zebrafish possess two aanats, aanat1 and aanat2; the former is expressed only in the retina and the latter is expressed in both the retina and the pineal gland. Here, their differential expression and regulation were studied using transcript quantification and transient and stable in vivo and in vitro transfection assays. In the pineal gland, the aanat2 promoter exhibited circadian clock-controlled activity, as indicated by circadian rhythms of Enhanced green fluorescent protein (EGFP) mRNA in AANAT2:EGFP transgenic fish. In vivo transient expression analyses of the aanat2 promoter indicated that E-box and photoreceptor conserved elements (PCE) are required for expression in the pineal gland. In the retina, the expression of both genes was characterized by a robust circadian rhythm of their transcript levels. In constant darkness, the rhythmic expression of retinal aanat2 persisted while the aanat1 rhythm disappeared; indicating that the former is controlled by a circadian clock and the latter is also light driven. In the light-entrainable clock-containing PAC-2 zebrafish cell line, both stably transfected aanat1 and aanat2 promoters exhibited a clock-controlled circadian rhythm, characteristic for an E-box-driven expression. Transient co-transfection experiments in NIH-3T3 cells revealed that the two, E-box- and PCE-containing, promoters are driven by the synergistic action of BMAL/CLOCK and orthehodenticle homeobox 5. This study has revealed a shared mechanism for the regulation of two related genes, yet describes their differential phases and photic responses which may be driven by other gene-specific regulatory mechanisms and tissue-specific transcription factor profiles.

Achilles-Mediated and Sex-Specific Regulation of Circadian mRNA Rhythms in Drosophila

2019 ◽  
Vol 34 (2) ◽  
pp. 131-143 ◽  
Author(s):  
Jiajia Li ◽  
Renee Yin Yu ◽  
Farida Emran ◽  
Brian E. Chen ◽  
Michael E. Hughes
Keyword(s):  
Circadian Clock ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Clock Genes ◽  
Peripheral Tissues ◽  
Knock Down ◽  
Rhythmic Expression ◽  
The Core ◽  
Physiological Processes ◽  
Specific Regulation

The circadian clock is an evolutionarily conserved mechanism that generates the rhythmic expression of downstream genes. The core circadian clock drives the expression of clock-controlled genes, which in turn play critical roles in carrying out many rhythmic physiological processes. Nevertheless, the molecular mechanisms by which clock output genes orchestrate rhythmic signals from the brain to peripheral tissues are largely unknown. Here we explored the role of one rhythmic gene, Achilles, in regulating the rhythmic transcriptome in the fly head. Achilles is a clock-controlled gene in Drosophila that encodes a putative RNA-binding protein. Achilles expression is found in neurons throughout the fly brain using fluorescence in situ hybridization (FISH), and legacy data suggest it is not expressed in core clock neurons. Together, these observations argue against a role for Achilles in regulating the core clock. To assess its impact on circadian mRNA rhythms, we performed RNA sequencing (RNAseq) to compare the rhythmic transcriptomes of control flies and those with diminished Achilles expression in all neurons. Consistent with previous studies, we observe dramatic upregulation of immune response genes upon knock-down of Achilles. Furthermore, many circadian mRNAs lose their rhythmicity in Achilles knock-down flies, suggesting that a subset of the rhythmic transcriptome is regulated either directly or indirectly by Achilles. These Achilles-mediated rhythms are observed in genes involved in immune function and in neuronal signaling, including Prosap, Nemy and Jhl-21. A comparison of RNAseq data from control flies reveals that only 42.7% of clock-controlled genes in the fly brain are rhythmic in both males and females. As mRNA rhythms of core clock genes are largely invariant between the sexes, this observation suggests that sex-specific mechanisms are an important, and heretofore under-appreciated, regulator of the rhythmic transcriptome.

scholarly journals Single-cell analysis of circadian dynamics in tissue explants

2015 ◽  
Vol 26 (22) ◽  
pp. 3940-3945 ◽  
Author(s):  
Laura Lande-Diner ◽  
Jacob Stewart-Ornstein ◽  
Charles J. Weitz ◽  
Galit Lahav
Keyword(s):  
Circadian Clock ◽  
Single Cell ◽  
Cell Tracking ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Tissue Imaging ◽  
Wide Field ◽  
Isolated Cells ◽  
Peripheral Tissues

Tracking molecular dynamics in single cells in vivo is instrumental to understanding how cells act and interact in tissues. Current tissue imaging approaches focus on short-term observation and typically nonendogenous or implanted samples. Here we develop an experimental and computational setup that allows for single-cell tracking of a transcriptional reporter over a period of >1 wk in the context of an intact tissue. We focus on the peripheral circadian clock as a model system and measure the circadian signaling of hundreds of cells from two tissues. The circadian clock is an autonomous oscillator whose behavior is well described in isolated cells, but in situ analysis of circadian signaling in single cells of peripheral tissues is as-yet uncharacterized. Our approach allowed us to investigate the oscillatory properties of individual clocks, determine how these properties are maintained among different cells, and assess how they compare to the population rhythm. These experiments, using a wide-field microscope, a previously generated reporter mouse, and custom software to track cells over days, suggest how many signaling pathways might be quantitatively characterized in explant models.

Zebrafish circadian clocks: cells that see light

2005 ◽  
Vol 33 (5) ◽  
pp. 962-966 ◽  
Author(s):  
T.K. Tamai ◽  
A.J. Carr ◽  
D. Whitmore
Keyword(s):  
Circadian Clock ◽  
Cell Lines ◽  
Early Stage ◽  
Imaging Techniques ◽  
Light Sensitivity ◽  
Gene Promoters ◽  
Peripheral Tissues ◽  
Mammalian Cell Lines ◽  
Direct Light ◽  
Daily Rhythmicity

In the classical view of circadian clock organization, the daily rhythms of most organisms were thought to be regulated by a central, ‘master’ pacemaker, usually located within neural structures of the animal. However, with the results of experiments performed in zebrafish, mammalian cell lines and, more recently, mammalian tissues, this view has changed to one where clock organization is now seen as being highly decentralized. It is clear that clocks exist in the peripheral tissues of animals as diverse as Drosophila, zebrafish and mammals. In the case of Drosophila and zebrafish, these tissues are also directly light-responsive. This light sensitivity and direct clock entrainability is also true for zebrafish cell lines and early-stage embryos. Using luminescent reporter cell lines containing clock gene promoters driving the expression of luciferase and single-cell imaging techniques, we have been able to show how each cell responds rapidly to a single light pulse by being shifted to a common phase, equivalent to the early day. This direct light sensitivity might be related to the requirement for light in these cells to activate the transcription of genes involved in DNA repair. It is also clear that the circadian clock in zebrafish regulates the timing of the cell cycle, demonstrating the wide impact that this light sensitivity and daily rhythmicity has on the biology of zebrafish.

scholarly journals Identification of PCBP1 as a Novel Modulator of Mammalian Circadian Clock

2021 ◽  
Vol 12 ◽  
Author(s):  
Yaling Wu ◽  
Haijiao Zhao ◽  
Eric Erquan Zhang ◽  
Na Liu
Keyword(s):  
Circadian Clock ◽  
Time Course ◽  
Binding Protein ◽  
Mechanistic Study ◽  
Daily Cycle ◽  
Rhythmic Expression ◽  
Circadian Expression ◽  
E Box ◽  
Cryptochrome 1

The circadian clock governs our daily cycle of behavior and physiology. Previous studies have identified a handful of core clock components and hundreds of circadian modifiers. Here, we report the discovery that poly(C)-binding protein 1 (PCBP1), displaying a circadian expression pattern, was a novel circadian clock regulator. We found that knocking down PCBP1 resulted in period shortening in human U2OS cells, and that manipulations of PCBP1 expression altered the activity of CLOCK/BMAL1 in an E-box-based reporter assay. Further mechanistic study demonstrated that this clock function of PCBP1 appears to work by enhancing the association of Cryptochrome 1 (CRY1) with the CLOCK/BMAL1 complex, thereby negatively regulating the latter’s activation. Co-immunoprecipitation of PCBP1 and core clock molecules confirmed the interactions between PCBP1 and CRY1, and a time-course qPCR assay revealed the rhythmic expression of PCBP1 in mouse hearts in vivo. Given that the RNA interference of mushroom-body expressed (mub), the poly(rC) binding protein (PCBP) homolog of Drosophila, in the clock neurons also led to a circadian phenotype in the locomotor assay, our study deemed PCBP1 a novel clock modifier whose circadian regulatory mechanism is conserved during evolution.

Circadian rhythm of acidification in insect vas deferens regulated by rhythmic expression of vacuolar H+-ATPase

2002 ◽  
Vol 205 (1) ◽  
pp. 37-44
Author(s):  
Piotr Bebas ◽  
Bronislaw Cymborowski ◽  
Jadwiga M. Giebultowicz
Keyword(s):  
Circadian Rhythm ◽  
Circadian Clock ◽  
Vas Deferens ◽  
Apical Cell ◽  
Transport Processes ◽  
Daily Rhythm ◽  
Constant Darkness ◽  
Cotton Leaf ◽  
Apical Cell Membrane ◽  
Peripheral Tissues

SUMMARY Recent studies have demonstrated that the peripheral tissues of vertebrates and invertebrates contain circadian clocks; however, little is known about their functions and the rhythmic outputs that they generate. To understand clock-controlled rhythms at the cellular level, we investigated a circadian clock located in the reproductive system of a male moth (the cotton leaf worm Spodoptera littoralis) that is essential for the production of fertile spermatozoa. Previous work has demonstrated that spermatozoa are released from the testes in a daily rhythm and are periodically stored in the upper vas deferens (UVD). In this paper, we demonstrate a circadian rhythm in pH in the lumen of the UVD, with acidification occurring during accumulation of spermatozoa in the lumen. The daily rhythm in pH correlates with a rhythmic increase in the expression of a proton pump, the vacuolar H+-ATPase (V-ATPase), in the apical portion of the UVD epithelium. Rhythms in pH and V-ATPase persist in light/dark cycles and constant darkness, but are abolished in constant light, a condition that disrupts clock function and renders spermatozoa infertile. Treatment with colchicine impairs the migration of V-ATPase-positive vesicles to the apical cell membrane and abates the acidification of the UVD lumen. Bafilomycin, a selective inhibitor of V-ATPase activity, also prevents the decline in luminal pH. We conclude that the circadian clock generates a rhythm of luminal acidification by regulating the levels and subcellular distribution of V-ATPase in the UVD epithelium. Our data provide the first evidence for circadian control of V-ATPase, the fundamental enzyme that provides the driving force for numerous secondary transport processes. They also demonstrate how circadian rhythms displayed by individual cells contribute to the synchrony of physiological processes at the organ level.

scholarly journals Transcriptional Structure of Petunia Clock in Leaves and Petals

Genes ◽  
2019 ◽  
Vol 10 (11) ◽  
pp. 860 ◽  
Author(s):  
Marta I. Terry ◽  
Marta Carrera-Alesina ◽  
Julia Weiss ◽  
Marcos Egea-Cortines
Keyword(s):  
Circadian Clock ◽  
Clock Genes ◽  
Time Of Day ◽  
Constant Darkness ◽  
Petunia X Hybrida ◽  
Environmental Signals ◽  
Expression Variability ◽  
Rhythmic Expression ◽  
Free Running ◽  
Maximum Expression

The plant circadian clock coordinates environmental signals with internal processes including secondary metabolism, growth, flowering, and volatile emission. Plant tissues are specialized in different functions, and petals conceal the sexual organs while attracting pollinators. Here we analyzed the transcriptional structure of the petunia (Petunia x hybrida) circadian clock in leaves and petals. We recorded the expression of 13 clock genes in petunia under light:dark (LD) and constant darkness (DD). Under light:dark conditions, clock genes reached maximum expression during the light phase in leaves and the dark period in petals. Under free running conditions of constant darkness, maximum expression was delayed, especially in petals. Interestingly, the rhythmic expression pattern of PhLHY persisted in leaves and petals in LD and DD. Gene expression variability differed among leaves and petals, time of day and photoperiod. The transcriptional noise was higher especially in leaves under constant darkness. We found that PhPRR7, PhPRR5, and PhGI paralogs showed changes in gene structure including exon number and deletions of CCT domain of the PRR family. Our results revealed that petunia petals presented a specialized clock.

scholarly journals Circadian control of the secretory pathway is a central mechanism in tissue homeostasis

2018 ◽  
Author(s):  
Joan Chang ◽  
Richa Garva ◽  
Adam Pickard ◽  
Ching-Yan Chloé Yeung ◽  
Venkatesh Mallikarjun ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Collagen Synthesis ◽  
Secretory Pathway ◽  
Protein Homeostasis ◽  
Collagen Network ◽  
Rhythmic Expression ◽  
Circadian Control ◽  
Collagen Accumulation ◽  
Clock Mechanism

Collagen is the most abundant secreted protein in vertebrates that persists throughout life without renewal. The unchanging nature of collagen contrasts with observed continued collagen synthesis throughout adulthood and with conventional transcriptional and translational homeostatic mechanisms that replace damaged proteins with new copies. Here we show circadian clock regulation of procollagen transport from ER-to-Golgi and Golgi-to-plasma membrane by sequential rhythmic expression of SEC61, TANGO1, PDE4D and VPS33B. The result is nocturnal procollagen synthesis and daytime collagen fibril assembly in mice. Rhythmic collagen degradation by CTSK maintains collagen homeostasis. This circadian cycle of collagen synthesis, assembly and degradation affects only a pool of newly-synthesized collagen whilst maintaining the persistent collagen network. Disabling the circadian clock causes collagen accumulation and abnormal fibrils in vivo. In conclusion, our study has identified a circadian clock mechanism of protein homeostasis in which a sacrificial pool of collagen is synthesized and removed to maintain tissue function.

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