Interplay of developmental clock and extracellular environment in brain formation

金沢大学理工研究域生命理工学系本研究では、発生プロセスが発生時計のレジリエンスとロバストネスに及ぼす影響を明らかにする目的で、ゼブラフィッシュ分節時計の同期過程を解析した。Notchシグナルによる細胞間シグナル伝達を阻害する薬剤を処理し、細胞間の分節時計の同期を崩すと、欠陥のある体節が形成される。薬剤を除去してしばらくすると、正常な体節が再び作られる。これは分節時計の再同期が起きたためだと考えられてきたが、その詳細は不明であった。この過程を明らかにするために、昨年度は空間三次元の数理モデルを構築し解析を行った。その結果、再同期過程において渦状の位相パターンが組織後方に現れ、正常な体節が作られた後で再び欠陥のある体節を作ることを見出した。今年度は、構築した数理モデルを用い、薬剤除去後初めて正常な体節が作られるまでにかかる時間と、欠陥のある体節が再び現れなくなるまでにかかる時間が、発生ステージに応じてどのように変化するかを数理的に解析し、実験データとの比較を行った。発生とともに組織長は短くなり、また組織の伸長様式も変化する。数値計算によると、組織長の短縮は、欠陥のある体節が現れなくなるまでにかかる時間を短くする。また伸長様式の変化もこの時間に影響を及ぼす。一方、正常な体節が初めて現れるまでにかかる時間は、Notchシグナルが分節時計に影響を及ぼす強さに大きく依存することが分かった。まとめると、隣接細胞間相互作用によって生じる渦状位相パターンが、正常な体節ができた後に再び欠陥のある体節が形成される現象を引き起こす。この渦状位相パターンの動きは組織長や組織伸長によって決まるため、これらの組織パラメタは再同期過程に影響を及ぼしうる。本研究の結果は組織伸長や組織サイズの変化といった発生プロセスが、発生時計の摂動応答に影響を及ぼすことを示唆する。研究課題/領域番号:17H05762, 研究期間(年度):2017-04-01 – 2019-03-3

概日時計におけるインターロック逆位相振動子の設計原理

In system biology, mathematical models have long tradition are used to understand complex biological control processes/ systems, for example, circadian clock oscillatory mechanism. Circadian rhythms (~24 hour) is ubiquitous in almost the living species ranging from mammals to cyanobacteria shows the robustness of key oscillatory features such as the phase, period and amplitude against external and internal variations. These autonomous oscillations are formed by the complex interactions of the interactive molecules. A transcriptional-translational feedback loop is typically characterized as a common principle for this sustained oscillations. Recently studies, it has broadly been established that the robustness of biochemical oscillators, like the Drosophila circadian clocks, can be generated by interlocked transcriptional-translational feedback loops, where two negative feedback loops are coupled through mutual activations. The mechanisms by which such coupling protocols have survived out of many possible protocols remain to be revealed. To address this question, we investigated two distinct coupling protocols: activator-coupled oscillators (ACO) and repressor-coupled oscillators (RCO). We focused on the two coupling parameters: coupling dissociation constant and coupling time delay. Interestingly, the ACO was able to produce anti-phase or morning-evening cycles, whereas the RCO produced in-phase ones. Deterministic and stochastic analyses demonstrated that the anti-phase ACO provided greater fluctuations in amplitude not only with respect to changes in coupling parameters but also to random parameter perturbations than the in-phase RCO. Moreover, the ACO deteriorated the entrainability to the day-night master clock, whereas the RCO produced high entrainability. Considering that the real, interlocked feedback loops have evolved as the ACO, instead of the RCO, we first proposed a hypothesis that the morning-evening or anti-phase cycle is more essential for Drosophila than achieving the robustness and entrainability.九州工業大学博士学位論文 学位記番号:情工博甲第352号 学位授与年月日:令和2年9月25日1 BACKGROUND|2 THE DYNAMICS MODELS OF CIRCADIAN RHYTHMS|3 MODELING THE INTERLOCKED NEGATIVE FEEDBACK LOOPS|4 ROBUSTNESS OF THE INTERLOCKED CIRCADIAN OSCILLATORS|5 ENTRAINABILITY OF THE COUPLED OSCILLATORS|6 CONCLUSIONS AND FUTURE WORK九州工業大学令和2年

Genomic and Physiological Characterization of the Mutant time for coffee within the Arabidopsis thaliana Circadian Clock

ircadian clocks are internal timekeepers that provide organisms with a sense of time. These oscillators, which are entrained by external stimuli, predict the daily day/night transitions and have a periodicity of about 24 hours. The Arabidopsis thaliana circadian clock is composed of interconnected transcriptional-translational feedback loops. The morning expressed elements CCA1 and LHY, which are clock controlled and light inducible, repress the transcription of the evening element TOC1. At dusk, TOC1 repression is relieved as CCA1 and LHY protein diminish. Then TOC1 stimulates the expression of the morning components closing the loop. Still, how the light signal at dawn entrains this clock has remained elusive. time for coffee (tic) was originally reported as a circadian-clock mutant based on its early phase and a short period. It was found that tic is defective in sensing dawn because its clock incorrectly resets before morning light. Because TIC mRNA and protein were found to be constant through a diurnal cycle, an activation event could trigger TIC time-specific function within the oscillator. Therefore TIC action takes place before the expression of CCA1 and LHY and coincides in time with clock entrainment by light. In this thesis, I report the results from a microarray study that led to a detailed phenotypic analysis of tic in an effort to uncover the mechanism to clock entrainment. I could confirm and expand the defective clock-gene expression profile of tic. Interestingly tic showed increased transcriptional changes in response to the environment compared to wild type. Global transcriptomic analysis indicated that tic has altered redox homeostasis and defects in ABA signalling pathways. Furthermore GO enrichment analysis highlighted that stress and environmental responses were among the most abundant categories misexpressed in tic. In conclusion, TIC was found to be an essential component for global transcriptome reprogramming to a dawn light signal. The results obtained through the microarray analysis directed me to demonstrate that tic resulted in an array of pleiotropic phenotypes. Besides its clock defects, tic presented hypersensitivity to oxidative stress, altered ABA-related signalling and responses, such as drought tolerance, defects in iron homeostasis, alterations in starch metabolism and disrupted stress responses. Furthermore it is suggested that tic has a role in nucleotide and secondary metabolism. All together, I concluded that TIC functions in maintaining metabolic homeostasis through modulation of stress responses. In summary from the data presented here, I hypothesize that clock entrainment occurs through metabolic signals, probably derived from photosynthesis and cellular energy homeostasis. These signals would be integrated to the oscillator by TIC. In this way, TIC would promote the anticipation of the oncoming new day

Phytochrome genes in higher plants: Structure,expression, and evolution

© 2006 Springer. All Rights Reserved. Phytochromes play critical roles in monitoring light quantity, quality, and periodicity in plants and they relay this photosensory information to a large number of signaling pathways that regulate plant growth and development. Given these complex functions, it is not surprising that the phytochrome apoproteins are encoded by small multigene families and that different forms of phytochrome regulate different aspects of photomorphogenesis. Over the course of the last decade, progress has been made in defining the number, molecular properties, and biological activities of the photoreceptors that constitute a plant R/FR sensing system. This chapter summarizes our current understanding of the structure of the genes that encode the phytochrome apoproteins (the PHY genes), the expression patterns of those genes, the nature of the phytochrome apoprotein family, and PHY gene evolution in seed plants. Phytochrome was discovered and its basic photochemical properties were first described through physiological studies of light-sensitive seed germination and photoperiodic effects on flowering (Borthwick, et al., 1948, Borthwick, et al., 1952). The pigment itself was initially isolated from extracts of dark-grown (etiolated) plant tissue in 1959 (Butler, et al., 1959), but it was not until much later that phytochrome was purified to homogeneity in an undegraded form (Vierstra and Quail, 1983). DNA sequences of gene and cDNA clones for oat etiolated-tissue spectroscopically in planta and purified in its native form, this dark-tissue phytochrome (now called phyA) remains the most completely biochemically and spectroscopically characterized form of the receptor. At various times throughout the first 40 years of the study of the abundant etiolated-tissue phytochrome, evidence for the presence and activity of additional forms of phytochrome, often referred to as green-tissue or light-stable phytochromes, was obtained. Initially, in physiological experiments, it was sometimes not possible to correlate specific in vivo phytochrome activities with the phytochrome provided the first complete descriptions of the apoprotein (Hershey et al., 1985). Because it accumulates to levels that permit it to be assayed known spectroscopic properties of the molecule. Later, direct evidence for multiple species of phytochrome in plants and in plant extracts was obtained using both spectroscopic and immunochemical methods (reviewed in Pratt, 1995). The molecular identities of these additional phytochrome forms were ultimately deduced from cDNA clones that were isolated by nucleic acid similarity to etiolated-tissue phytochrome sequences (Sharrock and Quail, 1989). More recently, analysis of a large number of complete and partial PHY gene or cDNA sequences from a broad sampling of plant phylogenetic groups and sequencing of several plant genomes have resulted in a much clearer and more general picture of what constitutes a higher plant R/FR photoreceptor family. It is likely that the major types of long-wavelength photosensing pigments have now been identified and the challenge that lies ahead is to understand how the signalling mechanisms, expression patterns, and interactions of these molecules contribute to plant responses to the R/FR environment. Extending the investigation of phytochrome gene families and their functions to additional angiosperm and gymnosperm genera will be an integral component of this effort and of our ability to utilize this growing understanding of phytochrome function to modify the agricultural properties of plants and to better understand the history of land plants

The role of photoperiod in the entrainment of endogenous clocks and rhythms in Antarctic krill (Euphausia superba)

Antarctic krill (Euphausia superba), hereafter krill, are key players in the ecosystem of the Southern Ocean. They are distributed all around Antarctica, and they are exceptionally abundant, representing the main link between primary producers and the higher trophic levels in the Antarctic marine food web. Due to their high ecological relevance, krill have been extensively studied in the field and in the laboratory, and it is known that their life-cycle is shaped by fundamental daily and seasonal rhythmic events. Actual knowledge about the external and internal factors involved in the regulation of rhythmic functions in krill is still quite limited but pivotal, especially in the context of future environmental changes driven by climate change. One hypothesis is that the daily and seasonal rhythmic functions in krill might be regulated through the activity of so-called “endogenous” clocks. Endogenous clocks are molecular function units, which promote rhythmic oscillations in transcription, physiology and behavior at the daily and seasonal levels. Endogenous clocks can be entrained (i.e. synchronized) by rhythmic environmental cues, like the day/night cycle (i.e. photoperiod = day length) at the daily level, and the seasonal photoperiodic cycle at the seasonal level. The implications of endogenous rhythmicity (i.e. rhythmicity promoted by endogenous clocks) in the regulation of rhythmic biological functions are well documented among terrestrial species, but studies dealing with marine organisms are very scarce. At the daily level, the best studied endogenous clock is the circadian clock, which is based on molecular feedback loops generating a rhythm with a period of approximately 24 h. Specific light-sensitive proteins promote the entrainment of the circadian clock with the day/night cycle, ensuring effective synchronization of rhythmic output functions according to daily recurring environmental changes. In krill, a circadian clock has been recently identified and characterized, and its influence on daily rhythms of metabolism and transcription has been demonstrated in the laboratory and in natural conditions. At the seasonal level, the regulation of rhythmic functions is less well understood, also in terrestrial species. An endogenous circannual clock seems to be involved, but the molecular mechanisms underlying its functioning are still unclear. Due to its ability to measure changes in day length, the circadian clock might contribute to the seasonal entrainment of the circannual clock. In krill, a circannual rhythm (i.e. a rhythm promoted by a circannual clock) might be involved in the regulation of the seasonal shifts in sexual maturity and metabolic activity observed in the field in summer and winter. During this dissertation, I investigated the involvement of endogenous clocks and rhythms in the regulation of rhythmic functions in krill at the daily and seasonal levels. Moreover, I also examined the role played by photoperiod in the entrainment of those clocks and rhythms. The work focused on three main research topics, which resulted in three publications: 1) the impact of the extreme seasonal photoperiodic cycle of the Southern Ocean on the activity of the circadian clock of krill at different times of the year (Publication I); 2) the involvement of an endogenous circannual rhythm and the role played by photoperiod in the regulation of the seasonal metabolic activity cycle of krill (Publication II); and 3) the involvement of the circadian clock and the role played by photoperiod in the regulation of diel vertical migration (DVM) in krill (Publication III). In publication I, I investigated the activity of the circadian clock of krill in different simulated seasonal Antarctic light conditions. The extreme variability displayed by the seasonal photoperiodic cycle in the Southern Ocean might cause a problem for the photoperiodic entrainment of the clock in different seasons. Especially during summer and winter, when overt light/dark cues are missing, the clock might get disrupted and the clock output might become arrhythmic. Indeed, laboratory work demonstrated that under simulated mid-summer and mid-winter conditions, when overt photoperiodic cues were missing, the circadian clock of krill was arrhythmic, and the metabolic output was de-synchronized. Conversely, under simulated early-autumn and late-winter conditions, when overt photoperiodic cues were present, the circadian clock of krill was active, and the metabolic output was synchronized with the light/dark cycle. This suggested that major changes are occurring during the year in the entraining process of the circadian clock of krill, depending on the different seasonal light conditions to which krill are exposed. In publication II, I investigated the involvement of an endogenous circannual rhythm in the regulation of the seasonal metabolic activity cycle of krill. Moreover, I also examined the role played by photoperiod in the entrainment of this rhythm. In response to the strong seasonal variability displayed by light and food availability in the Southern Ocean, krill display seasonal differences in metabolic rates, feeding activity and growth. During summer, when light and food availability is high, krill metabolic and feeding activity is enhanced, and krill growth rates are positive. During winter, when light and food conditions are low, krill metabolic and feeding activity is reduced, and krill show reduced growth or even shrinkage (i.e. reduction of size). It has been hypothesized that an endogenous rhythm entrained by the seasonal Antarctic light regime might be responsible for the regulation of the seasonal metabolic cycle of krill. Krill exposed to different long-term simulated natural seasonal light conditions, showed seasonal patterns of growth, enzyme activity and gene expression of key metabolic genes, which were also observed in krill exposed to constant darkness. The results strongly suggested the involvement of a circannual clock in the regulation of the seasonal metabolic cycle of krill. However, major differences were observed in the seasonal patterns of oxygen consumption, suggesting that exposition of krill to specific seasonal light cues might be necessary for the effective entrainment of the circannual clock. In publication III, I investigated the involvement of an endogenous circadian rhythm in the regulation of krill diel vertical migration (DVM). Moreover, I also examined the role played by photoperiod in the entrainment of krill DVM. DVM is a mass migratory movement displayed by many zooplankton species worldwide. During the night, the animals come to the surface to graze on phytoplankton, while during the day they sink to deeper layers to escape from visual predators. The environmental factors involved in the regulation of DVM are photoperiod, food availability and presence/absence of predators. However, DVM occurs also in constantly dark environments (e.g. the deep sea and the Arctic ocean during the polar night), suggesting the involvement of an endogenous rhythm of regulation. Using krill exposed to different light/dark (LD) and constant darkness (DD) conditions, I found that krill DVM was driven by an endogenous rhythm, with krill moving upward during the light phase and downward during the dark phase. A similar rhythm was found in krill oxygen consumption, confirming the presence of an endogenous rhythm of activity associated with DVM. Rhythmic expression of clock genes related to the circadian clock was found in the eyestalks of krill entrained to similar LD conditions, suggesting that an involvement of the circadian clock in the regulation of krill DVM would be possible. Major differences were observed among individual krill in the rhythmic regulation of DVM and oxygen consumption, suggesting that the circadian system of krill might display high degrees of individual plasticity. In conclusion, this dissertation improves our knowledge about the mechanisms regulating daily and seasonal rhythmic functions in the Antarctic krill, E. superba. The implication of endogenous rhythmicity was demonstrated for krill DVM at the daily level, and for krill seasonal metabolic cycle at the seasonal level. Photoperiod proved to be a most fundamental factor for the entrainment of krill DVM and krill seasonal metabolic cycle, as well as for the modulation of the activity of the circadian clock of krill at different times of the year. This work provides an example of how techniques which have been developed to study the molecular biology and chronobiology of terrestrial model species can be applied to the study of ecologically relevant species in the marine environments. In the future, understanding the regulation of rhythmic functions in ecological key marine species like Antarctic krill will help us to understand how these species will adapt to environmental changes driven by climate change

Plant Physiology, Development and Metabolism

Water is one of the most important constituents of life. Chemically, water is the hydride of oxygen. Oxygen, being more electronegative, exerts a strong attractive pull on its electrons. This unequal attraction results in small positive charge on twohydrogenmoleculesandasmallnegativechargeontheoxygenmolecule.The two lone pairs of electrons of the oxygen molecule result in bending of water molecule. The partial charges on oxygen and hydrogen molecules result in high electric dipole moment and polarity of water molecule

Circadian clocks, glucocorticoids and the gated inflammatory response

In mammals endogenous, self sustained oscillators, known as circadian clocks, have evolved as a result of day night cycles, with a period close to 24 hours, and are involved in many physiological processes; such as sleep wake cycles, metabolic and hormonal activity. The suprachiasmatic nucleus (SCN), is the central oscillator, and is synchronised to the external environment by light, via the eye. It has been demonstrated that peripheral clocks, too, contain the circadian oscillator, with tissues such as the lung, liver, heart and kidney as well as many isolated cell types remaining rhythmic, in culture, for many days. However, these peripheral oscillators require a signal from the central oscillator in order to co-ordinate a synchronised time. Leading candidates in the relay of this information are the circulating glucocorticoid hormones corticosterone (rodents) or cortisol (man), which are known to have potent effects on the peripheral clock, both in-vivo and in-vitro. Further to this, glucocorticoids have been used for many decades to suppress the symptoms of inflammation, a by product of many human diseases.This thesis aims to address the temporal regulation of the peripheral clock by the endogenous glucocorticoid, corticosterone, using a transgenic mouse harbouring a luciferase conjugated clock reporter, and circadian reporter cell lines. It also aims to address the relative contribution of the two closely related nuclear hormone receptors, the glucocorticoid and mineralocorticoid receptors. A further aim of the work with glucocorticoid signalling was to design a flow-though culture system, in order to address the effects of the endogenous pulsatile release of glucocorticoids on the peripheral oscillator. This thesis also aims to characterise the inflammatory response in relation to its circadian characteristics; its relationship with corticosterone and the effect of inflammation on the central clock components. Finally, this thesis aims to investigate a potential input/output of the clock, a member of the family of C/EBP transcription factors, C/EBP alpha, and whether it is under endogenous circadian control and regulated by glucocorticoids.Work in this thesis has shown that glucocorticoids dynamically regulate the peripheral clock at all phases of the circadian cycle and that this regulation occurs mainly through the glucocorticoid receptor; yet the mineralocorticoid receptor does have a function in the immediate response to glucocorticoid administration. Furthermore, as a result of the initial temporal profile after corticosterone addition, on the clock protein PERIOD2, I have shown transient regulation of the clock through Caveolin-1 based signalling. There is also a significant circadian component to the inflammatory response, which appears, at least in part, to be REV-ERB alpha mediated, and the inflammatory response also has profound effects on circadian gene expression in the periphery. A functional flow-through system was designed and a working model produced, albeit with technical difficulties, to address glucocorticoid pulsing and circadian timing but much more work is needed for effects to be fully understood. C/EBP alpha appears not to be under circadian regulation nor under direct glucocorticoid regulation, at least in peripheral models used here.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Tree Peony Species Are a Novel Resource for Production of α-Linolenic Acid

Tree peony is known worldwide for its excellent ornamental and medical values, but recent reports that their seeds contain over 40% α-linolenic acid (ALA), an essential fatty acid for humans drew additional interest of biochemists. To understand the key factors that contribute to this rich accumulation of ALA, we carried out a comprehensive study of oil accumulation in developing seeds of nine wild tree peony species. The fatty acid content and composition was highly variable among the nine species; however, we selected a high- (P. rockii) and low-oil (P. lutea) accumulating species for a comparative transcriptome analysis. Similar to other oilseed transcriptomic studies, upregulation of select genes involved in plastidial fatty acid synthesis, and acyl editing, desaturation and triacylglycerol assembly in the endoplasmic reticulum was noted in seeds of P. rockii relative to P. lutea. Also, in association with the ALA content, transcript levels for fatty acid desaturases (SAD, FAD2 and FAD3), which encode for enzymes necessary for polyunsaturated fatty acid synthesis were higher in P. rockii compared to P. lutea. We further showed that the overexpression of PrFAD2 and PrFAD3 in Arabidopsis increased linoleic and α-linolenic acid content, respectively and modulated their final ratio in the seed oil. In conclusion, we identified the key steps that contribute to efficient ALA synthesis and validated the necessary desaturases in P. rockii that are responsible for not only increasing oil content but also modulating 18:2/18:3 ratio in seeds. Together, these results will aid to improve essential fatty acid content in seeds of tree peonies and other crops of agronomic interest

UVR8 mediated spatial differences as a prerequisite for UV-B induced inflorescence phototropism

In Arabidopsis hypocotyls, phototropins are the dominant photoreceptors for the positive phototropism response towards unilateral ultraviolet-B (UV-B) radiation. We report a stark contrast of response mechanism with inflorescence stems with a central role for UV RESISTANCE LOCUS 8 (UVR8). The perception of UV-B occurs mainly in the epidermis and cortex with a lesser contribution of the endodermis. Unilateral UV-B exposure does not lead to a spatial difference in UVR8 protein levels but does cause differential UVR8 signal throughout the stem with at the irradiated side 1) increase of the transcription factor ELONGATED HYPOCOTYL 5 (HY5), 2) an associated strong activation of flavonoid biosynthesis genes and flavonoid accumulation, 3) increased GA2oxidase expression, diminished gibberellin1 levels and accumulation of DELLA protein REPRESSOR OF GA1 (RGA) and, 4) increased expression of the auxin transport regulator, PINOID, contributing to local diminished auxin signalling. Our molecular findings are in support of the Blaauw theory (1919), suggesting that differential growth occurs trough unilateral photomorphogenic growth inhibition. Together the data indicate phototropin independent inflorescence phototropism through multiple locally UVR8-regulated hormone pathways