Project description:In Arabidopsis, the root clock regulates the spacing of lateral organs along the primary root through oscillating gene expression. The core molecular mechanism that drives the root clock periodicity and how it is modified by exogenous cues such as auxin and gravity remain unknown. We identified the key elements of the oscillator (AUXIN RESPONSE FACTOR 7, its auxin-sensitive inhibitor IAA18/POTENT, and auxin) that form a negative regulatory loop circuit in the oscillation zone. Through multilevel computer modeling fitted to experimental data, we explain how gene expression oscillations coordinate with cell division and growth to create the periodic pattern of organ spacing. Furthermore, gravistimulation experiments based on the model predictions show that external auxin stimuli can lead to entrainment of the root clock. Our work demonstrates the mechanism underlying a robust biological clock and how it can respond to external stimuli.
Project description:Oscillatory lysosomal activity for disposal of the components of the cellular clock is essential to sustain organismal circadian rhythms
Project description:Wild tobacco flowers wave rhythmically to facilitate specific pollinator interactions. This movement behavior is controlled by a regulatory network that involves the circadian clock- and auxin-signaling pathways. The plant hormone auxin, similarly to its function in tropic movements, acts as growth regulator in the circadian regulation of floral movement. Dorsoventral asymmetry in auxin levels and auxin transcriptional responses mediate the growth responses in the floral peduncle that make flowers move. Multiple components of the auxin-signaling pathway and auxin responses are under the control of circadian clock. However, it is unclear where these two pathways intersect and how collectively contribute to regulate specific rhythmic outputs. Here we found that the blue light photoreceptor and circadian clock component ZEITLUPE (ZTL) controls auxin responses through the regulation of the auxin-signaling pathway in a time-of-day and blue light specific manner. Abrogation of ZTL expression abolishes flower movement and the temporal gating of auxin-induced growth responses in the floral peduncle. ZTL regulates transcription and directly interacts with indole-3-acetic acid inducible 19 (IAA19), a circadian controlled gene that regulates development of curvature in moving organs. Indicating that ZTL modulates auxin sensitivity in part through the regulation of AUX/IAA transcriptional repressors. At night, growth responses in the peduncle to the synthetic auxin 2,4-D revealed that ZTL additionally controls auxin responses regulating auxin homeostasis. These results indicate that ZTL conveys temporal and environmental information, at multiple levels, into the auxin signaling-pathway and in this way sculpts the temporal gating of auxin responses that allow flowers to move. To gain further insight into the molecular basis of temporal regulation of the movement of flowers we used a whole genome microarray as a discovery platform to identify genes differentially expressed in a RNAi knockdown line silenced in the expression of the circadian clock component ZEITLUPE (irZTL-314).
Project description:The liver circadian clock is reprogrammed by nutritional challenge through the rewiring of specific transcriptional pathways. As the gut microbiota is tightly connected to host metabolism, whose coordination is governed by the circadian clock, we explored whether gut microbes influence circadian homeostasis and how they distally control the peripheral clock in the liver. Using fecal transplant procedures we reveal that, in response to high fat diet, the gut microbiota drives PPARγ-mediated activation of newly oscillatory transcriptional programs in the liver. Moreover, antibiotics treatment prevents PPARγ-driven transcription in the liver, underscoring the essential role of gut microbes in clock reprogramming and hepatic circadian homeostasis. Thus, a specific molecular signature characterizes the influence of the gut microbiome in the liver, leading to the transcriptional rewiring of hepatic metabolism. We used microarray to quantify the tissue specific expression level of circadian genes in terms of total RNA.
Project description:Gene expression profiling of distinct members of a neuronal circuit has the potential to identify candidate molecules and mechanisms that underlie the formation, organization and function of the circuit. To this end, we report here the application of a novel method to characterize RNAs from small numbers of specific Drosophila brain neurons, which belong to the circadian circuit. We identified three different sets of mRNAs enriched in different subclasses of clock neurons: one is enriched in all clock neurons, a second is enriched in PDF-positive clock neurons and a third is enriched in PDF-negative clock neurons. Moreover, we characterized 2 novel genes, Fer2 and dnocturnin, one from each subgroup, which highlight subgroup-specific features and play important roles in circadian rhythms. The methodology is a powerful tool not only to dissect the cellular and molecular basis of circadian rhythms but also to molecularly characterize other Drosophila neuronal circuits. Experiment Overall Design: Circadican related neuronal celltypes (Tim, Pdf) or general neurons (Elav) were labeled by GFP or YFP using specific Gal4 drivers. Expression of those celltypes were profiled after manual sorting of those GFP or YFP positive cells. 3 biological replicates were collected (except adult small pdf cells).
Project description:During somitogenesis, oscillatory expression of genes in the notch and wnt signaling pathways plays a key role in regulating segmentation. These oscillations in expression levels are elements of a species-specific developmental mechanism. To date, the periodicity and components of the human clock remain unstudied. Here we show that a human mesenchymal stem/stromal cell (MSC) model can be induced to display oscillatory gene expression. We observed that the known cycling gene HES1 oscillated with a 5 hour period, consistent with available data on the rate of somitogenesis in humans. We also observed cycling of Hes1 expression in mouse C2C12 myoblasts with a period of 2 hours, consistent with previous in vitro and embryonic studies. Furthermore, we used microarray and quantitative PCR (Q-PCR) analysis to identify additional genes that display oscillatory expression both in vitro and in mouse embryos. We confirmed oscillatory expression of the notch pathway gene Maml3 and the wnt pathway gene Nkd2 by whole mount in situ hybridization analysis and Q-PCR. Expression patterns of these genes were disrupted in Wnt3atm1Amc mutants but not in Dll3pu mutants. Our results demonstrate that human and mouse in vitro models can recapitulate oscillatory expression observed in embryo and that a number of genes in multiple developmental pathways display dynamic expression in vitro. Keywords: time series
Project description:During somitogenesis, oscillatory expression of genes in the notch and wnt signaling pathways plays a key role in regulating segmentation. These oscillations in expression levels are elements of a species-specific developmental mechanism. To date, the periodicity and components of the human clock remain unstudied. Here we show that a human mesenchymal stem/stromal cell (MSC) model can be induced to display oscillatory gene expression. We observed that the known cycling gene HES1 oscillated with a 5 hour period, consistent with available data on the rate of somitogenesis in humans. We also observed cycling of Hes1 expression in mouse C2C12 myoblasts with a period of 2 hours, consistent with previous in vitro and embryonic studies. Furthermore, we used microarray and quantitative PCR (Q-PCR) analysis to identify additional genes that display oscillatory expression both in vitro and in mouse embryos. We confirmed oscillatory expression of the notch pathway gene Maml3 and the wnt pathway gene Nkd2 by whole mount in situ hybridization analysis and Q-PCR. Expression patterns of these genes were disrupted in Wnt3atm1Amc mutants but not in Dll3pu mutants. Our results demonstrate that human and mouse in vitro models can recapitulate oscillatory expression observed in embryo and that a number of genes in multiple developmental pathways display dynamic expression in vitro. Experiment Overall Design: Synchronization of C2C12 myoblasts was carried out as follows: T-25 cm flasks were set up in parallel. These cells were grown to 90% confluence in DMEM supplemented with 5% FBS then incubated in DMEM with only 0.2% FBS for 24 hours, and returned to DMEM supplemented with FBS. Samples were collected every 30 min. for 8 hours.
Project description:During somitogenesis, oscillatory expression of genes in the notch and wnt signaling pathways plays a key role in regulating segmentation. These oscillations in expression levels are elements of a species-specific developmental mechanism. To date, the periodicity and components of the human clock remain unstudied. Here we show that a human mesenchymal stem/stromal cell (MSC) model can be induced to display oscillatory gene expression. We observed that the known cycling gene HES1 oscillated with a 5 hour period, consistent with available data on the rate of somitogenesis in humans. We also observed cycling of Hes1 expression in mouse C2C12 myoblasts with a period of 2 hours, consistent with previous in vitro and embryonic studies. Furthermore, we used microarray and quantitative PCR (Q-PCR) analysis to identify additional genes that display oscillatory expression both in vitro and in mouse embryos. We confirmed oscillatory expression of the notch pathway gene Maml3 and the wnt pathway gene Nkd2 by whole mount in situ hybridization analysis and Q-PCR. Expression patterns of these genes were disrupted in Wnt3atm1Amc mutants but not in Dll3pu mutants. Our results demonstrate that human and mouse in vitro models can recapitulate oscillatory expression observed in embryo and that a number of genes in multiple developmental pathways display dynamic expression in vitro. Experiment Overall Design: Synchronization of human umbilical cord blood derived stem/stromal cell (population 1) was carried out as follows: T-25 cm flasks were set up in parallel. These cells were grown to 90% confluence in DMEM supplemented with 10% FBS (UCB-MSC), then incubated in DMEM with only 0.2% FBS for 24 hours, and returned to DMEM supplemented with FBS. Samples were collected at 30 min. intervals from 0 to 8 hours, and then at 1 hour intervals from 9 to 24 hours.