Thomas Dickmeis

Glucocorticoids and the circadian clock.

Glucocorticoids, hormones produced by the adrenal gland cortex, perform numerous functions in body homeostasis and the response of the organism to external stressors. One striking feature of their regulation is a diurnal release pattern, with peak levels linked to the start of the activity phase. This release is under control of the circadian clock, an endogenous biological timekeeper that acts to prepare the organism for daily changes in its environment. Circadian control of glucocorticoid production and secretion involves a central pacemaker in the hypothalamus, the suprachiasmatic nucleus, as well as a circadian clock in the adrenal gland itself. Central circadian regulation is mediated via the hypothalamic-pituitary-adrenal axis and the autonomic nervous system, while the adrenal gland clock appears to control sensitivity of the gland to the adrenocorticopic hormone (ACTH). The rhythmically released glucocorticoids in turn might contribute to synchronisation of the cell-autonomous clocks in the body and interact with them to time physiological dynamics in their target tissues around the day.

Temperature regulates transcription in the zebrafish circadian clock.

It has been well-documented that temperature influences key aspects of the circadian clock. Temperature cycles entrain the clock, while the period length of the circadian cycle is adjusted so that it remains relatively constant over a wide range of temperatures (temperature compensation). In vertebrates, the molecular basis of these properties is poorly understood. Here, using the zebrafish as an ectothermic model, we demonstrate first that in the absence of light, exposure of embryos and primary cell lines to temperature cycles entrains circadian rhythms of clock gene expression. Temperature steps drive changes in the basal expression of certain clock genes in a gene-specific manner, a mechanism potentially contributing to entrainment. In the case of the per4 gene, while E-box promoter elements mediate circadian clock regulation, they do not direct the temperature-driven changes in transcription. Second, by studying E-box-regulated transcription as a reporter of the core clock mechanism, we reveal that the zebrafish clock is temperature-compensated. In addition, temperature strongly influences the amplitude of circadian transcriptional rhythms during and following entrainment by light–dark cycles, a property that could confer temperature compensation. Finally, we show temperature-dependent changes in the expression levels, phosphorylation, and function of the clock protein, CLK. This suggests a mechanism that could account for changes in the amplitude of the E-box-directed rhythm. Together, our results imply that several key transcriptional regulatory elements at the core of the zebrafish clock respond to temperature.

Glucocorticoids Play a Key Role in Circadian Cell Cycle Rhythms

Clock output pathways play a pivotal role by relaying timing information from the circadian clock to a diversity of physiological systems. Both cell-autonomous and systemic mechanisms have been implicated as clock outputs; however, the relative importance and interplay between these mechanisms are poorly understood. The cell cycle represents a highly conserved regulatory target of the circadian timing system. Previously, we have demonstrated that in zebrafish, the circadian clock has the capacity to generate daily rhythms of S phase by a cell-autonomous mechanism in vitro. Here, by studying a panel of zebrafish mutants, we reveal that the pituitary–adrenal axis also plays an essential role in establishing these rhythms in the whole animal. Mutants with a reduction or a complete absence of corticotrope pituitary cells show attenuated cell-proliferation rhythms, whereas expression of circadian clock genes is not affected. We show that the corticotrope deficiency is associated with reduced cortisol levels, implicating glucocorticoids as a component of a systemic signaling pathway required for circadian cell cycle rhythmicity. Strikingly, high-amplitude rhythms can be rescued by exposing mutant larvae to a tonic concentration of a glucocorticoid agonist. Our work suggests that cell-autonomous clock mechanisms are not sufficient to establish circadian cell cycle rhythms at the whole-animal level. Instead, they act in concert with a systemic signaling environment of which glucocorticoids are an essential part.

The Light Responsive Transcriptome of the Zebrafish: Function and Regulation

Most organisms possess circadian clocks that are able to anticipate the day/night cycle and are reset or “entrained” by the ambient light. In the zebrafish, many organs and even cultured cell lines are directly light responsive, allowing for direct entrainment of the clock by light. Here, we have characterized light induced gene transcription in the zebrafish at several organizational levels. Larvae, heart organ cultures and cell cultures were exposed to 1- or 3-hour light pulses, and changes in gene expression were compared with controls kept in the dark. We identified 117 light regulated genes, with the majority being induced and some repressed by light. Cluster analysis groups the genes into five major classes that show regulation at all levels of organization or in different subset combinations. The regulated genes cover a variety of functions, and the analysis of gene ontology categories reveals an enrichment of genes involved in circadian rhythms, stress response and DNA repair, consistent with the exposure to visible wavelengths of light priming cells for UV-induced damage repair. Promoter analysis of the induced genes shows an enrichment of various short sequence motifs, including E- and D-box enhancers that have previously been implicated in light regulation of the zebrafish period2 gene. Heterologous reporter constructs with sequences matching these motifs reveal light regulation of D-box elements in both cells and larvae. Morpholino-mediated knock-down studies of two homologues of the D-box binding factor Tef indicate that these are differentially involved in the cell autonomous light induction in a gene-specific manner. These findings suggest that the mechanisms involved in period2 regulation might represent a more general pathway leading to light induced gene expression.

Nodal and Fgf pathways interact through a positive regulatory loop and synergize to maintain mesodermal cell populations

Interactions between Nodal/Activin and Fibroblast growth factor (Fgf) signalling pathways have long been thought to play an important role in mesoderm formation. However, the molecular and cellular processes underlying these interactions have remained elusive. Here, we address the epistatic relationships between Nodal and Fgf pathways during early embryogenesis in zebrafish. First, we find that Fgf signalling is required downstream of Nodal signals for inducing the Nodal co-factor One-eyed-pinhead (Oep). Thus, Fgf is likely to be involved in the amplification and propagation of Nodal signalling during early embryonic stages. This could account for the previously described ability of Fgf to render cells competent to respond to Nodal/Activin signals. In addition, overexpression data shows that Fgf8 and Fgf3 can take part in this process. Second, combining zygotic mutations in ace/fgf8 and oep disrupts mesoderm formation, a phenotype that is not produced by either mutation alone and is consistent with our model of an interdependence of Fgf8 and Nodal pathways through the genetic regulation of the Nodal co-factor Oep and the cell propagation of Nodal signalling. Moreover, mesodermal cell populations are affected differentially by double loss-of-function of Zoep;ace. Most of the dorsal mesoderm undergoes massive cell death by the end of gastrulation, in contrast to either single-mutant phenotype. However, some mesoderm cells are still able to undergo myogenic differentiation in the anterior trunk of Zoep;ace embryos, revealing a morphological transition at the level of somites 6-8. Further decreasing Oep levels by removing maternal oep products aggravates the mesodermal defects in double mutants by disrupting the fate of the entire mesoderm. Together, these results demonstrate synergy between oep and fgf8 that operates with regional differences and is involved in the induction, maintenance, movement and survival of mesodermal cell populations.

The circadian clock and glucocorticoids – Interactions across many time scales

Glucocorticoids are steroid hormones of the adrenal gland that are an integral component of the stress response and regulate many physiological processes, including metabolism and immune response. Their release into the blood is highly dynamic and occurs in about hourly pulses, the amplitude of which is modulated in a daytime dependent fashion. In addition, in many species seasonal changes in basal glucocorticoid levels have been reported. In their target tissues, glucocorticoids bind to cytoplasmic receptors of the nuclear receptor superfamily. Upon binding, these receptors regulate transcription in a highly dynamic fashion, which involves stochastic binding to regulatory DNA elements on a time scale of seconds and heat shock protein mediated receptor-ligand complex recycling within minutes. The glucocorticoid hormone system interacts with another highly dynamic system, the circadian clock. The circadian clock is an endogenous biological timing mechanism that allows organisms to anticipate regular daily changes in their environment. It regulates daily rhythms of glucocorticoid release by a variety of mechanisms, modulates glucocorticoid signaling and is itself influenced by glucocorticoids. Here, we discuss mechanisms, functions and interactions of the circadian and glucocorticoid systems across time scales ranging from seconds (DNA binding by transcriptional regulators) to years (seasonal rhythms).

Automated Processing of Zebrafish Imaging Data: A Survey

Due to the relative transparency of its embryos and larvae, the zebrafish is an ideal model organism for bioimaging approaches in vertebrates. Novel microscope technologies allow the imaging of developmental processes in unprecedented detail, and they enable the use of complex image-based read-outs for high-throughput/high-content screening. Such applications can easily generate Terabytes of image data, the handling and analysis of which becomes a major bottleneck in extracting the targeted information. Here, we describe the current state of the art in computational image analysis in the zebrafish system. We discuss the challenges encountered when handling high-content image data, especially with regard to data quality, annotation, and storage. We survey methods for preprocessing image data for further analysis, and describe selected examples of automated image analysis, including the tracking of cells during embryogenesis, heartbeat detection, identification of dead embryos, recognition of tissues and anatomical landmarks, and quantification of behavioral patterns of adult fish. We review recent examples for applications using such methods, such as the comprehensive analysis of cell lineages during early development, the generation of a three-dimensional brain atlas of zebrafish larvae, and high-throughput drug screens based on movement patterns. Finally, we identify future challenges for the zebrafish image analysis community, notably those concerning the compatibility of algorithms and data formats for the assembly of modular analysis pipelines.

A chemical screening system for glucocorticoid stress hormone signaling in an intact vertebrate.

Glucocorticoids, steroid hormones of the adrenal gland, are an integral part of the stress response and regulate glucose metabolism. Natural and synthetic glucocorticoids are widely used in anti-inflammatory therapy but can have severe side effects. In vivo tests are needed to identify novel glucocorticoids and to screen compounds for unwanted effects on glucocorticoid signaling. We created the Glucocorticoid Responsive In vivoZebrafish Luciferase activitY assay to monitor glucocorticoid signaling in vivo. The GRIZLY assay detects stress-induced glucocorticoid production in single zebrafish larvae, measures disruption of glucocorticoid signaling by an organotin pollutant metabolite, and specifically identifies a compound stimulating endogenous glucocorticoid production in a chemical screen. Our assay has broad applications in stress research, environmental monitoring, and drug discovery.

Start the clock! Circadian rhythms and development.

The contribution of timing cues from the environment to the coordination of early developmental processes is poorly understood. The day–night cycle represents one of the most important, regular environmental changes that animals are exposed to. A key adaptation that allows animals to anticipate daily environmental changes is the circadian clock. In this review, we aim to address when a light‐regulated circadian clock first emerges during development and what its functions are at this early stage. In particular, do circadian clocks regulate early developmental processes? We will focus on results obtained with Drosophila and vertebrates, where both circadian clock and developmental control mechanisms have been intensively studied. Developmental Dynamics 236:142–155, 2007.

Glucocorticoids and circadian clock control of cell proliferation: at the interface between three dynamic systems.

The circadian clock, an endogenous timekeeper that regulates daily rhythms of physiology, also influences the dynamic release of glucocorticoids. The release of glucocorticoids is characteristically pulsatile and is further modulated in a circadian fashion. A circadian pacemaker in the brain regulates daily rhythms of hypothalamic-pituitary-adrenal axis and autonomic nervous system activity that both influence glucocorticoid release from the adrenal gland. This systemic regulation interacts with rhythms in the adrenal gland itself that are driven by its own circadian clock. One function of glucocorticoids is the regulation of cell proliferation. Depending on the tissue, this can involve both negative and positive regulation of a variety of processes, including cell differentiation and cell death. Cell proliferation is also under circadian control, and recent evidence suggests that this regulation may involve glucocorticoid signalling. Here, we review the dynamic processes participating in the interplay between the circadian clock, glucocorticoids and cell proliferation, and we discuss the potential implications for therapy.