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.

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.

Altered daily patterns of hormone action are suspected to contribute to metabolic disease. It is poorly understood how the adrenal glucocorticoid hormones contribute to the coordination of daily global patterns of transcription and metabolism. Here, we examined diurnal metabolite and transcriptome patterns in a zebrafish glucocorticoid deficiency model by RNA-Seq, NMR spectroscopy and liquid chromatography-based methods. We observed dysregulation of metabolic pathways including glutaminolysis, the citrate and urea cycles and glyoxylate detoxification. Constant, non-rhythmic glucocorticoid treatment rescued many of these changes, with some notable exceptions among the amino acid related pathways. Surprisingly, the non-rhythmic glucocorticoid treatment rescued almost half of the entire dysregulated diurnal transcriptome patterns. A combination of E-box and glucocorticoid response elements is enriched in the rescued genes. This simple enhancer element combination is sufficient to drive rhythmic circadian reporter gene expression under non-rhythmic glucocorticoid exposure, revealing a permissive function for the hormones in glucocorticoid-dependent circadian transcription. Our work highlights metabolic pathways potentially contributing to morbidity in patients with glucocorticoid deficiency, even under glucocorticoid replacement therapy. Moreover, we provide mechanistic insight into the interaction between the circadian clock and glucocorticoids in the transcriptional regulation of metabolism. Glucocorticoids, steroid hormones of the adrenal gland, are important regulators of metabolism and the stress response. They are also widely used as anti-inflammatory drugs. Production and release of glucocorticoids show a diurnal pattern regulated by the circadian clock. Importantly, altered daily patterns of hormone action are thought to contribute to metabolic diseases. Here, we examined diurnal patterns of gene expression and metabolism in a zebrafish model of glucocorticoid deficiency. We observed that a surprisingly large number of genes show glucocorticoid-dependent diurnal patterns of transcription. This behaviour is particularly pronounced in metabolic genes, and metabolites of various central metabolic pathways are dysregulated. Interestingly, non-rhythmic glucocorticoid treatment restored many of these metabolite changes. It also restored expression of almost half of the dysregulated genes. Using in vivo bioluminescence assays, we provide evidence that this rescue is mediated via combined glucocorticoid–circadian clock activity on a simple regulatory DNA module enriched in the genomic vicinity of these genes. Our work provides mechanistic insight into how the circadian clock and glucocorticoids cooperate in the regulation of daily patterns of gene expression and metabolism. Furthermore, it highlights metabolic pathways that may contribute to disease mechanisms in patients with a disturbed glucocorticoid hormone system.

Cranial neural crest (NC) contributes to the developing vertebrate eye. By multidimensional, quantitative imaging, we traced the origin of the ocular NC cells to two distinct NC populations that differ in the maintenance of sox10 expression, Wnt signalling, origin, route, mode and destination of migration. The first NC population migrates to the proximal and the second NC cell group populates the distal (anterior) part of the eye. By analysing zebrafish pax6a/b compound mutants presenting anterior segment dysgenesis, we demonstrate that Pax6a/b guide the two NC populations to distinct proximodistal locations. We further provide evidence that the lens whose formation is pax6a/b-dependent and lens-derived TGFβ signals contribute to the building of the anterior segment. Taken together, our results reveal multiple roles of Pax6a/b in the control of NC cells during development of the anterior segment. Neural crest (NC) cells are pluripotent embryonic stem cells that originate from neural tissue during early embryogenesis. The origin and fate of NC cells have been studied intensively in various animal species showing that the eye receives input from NC cells. There are several inherited diseases like aniridia, Peters anomaly, Axenfeld-Rieger syndrome and congenital glaucoma affecting structures of the eye derived from NC cells. As of yet, it has remained unclear how the congenital abnormalities of the eye develop in these patients. We have applied genetic tools and multidimensional live imaging to study the dynamics of NC cell behaviour during eye formation. We found that two waves of NC cell populations with different properties contribute to distinct parts of the eye. By using zebrafish mutants as disease models for human anterior segment dysgenesis, we demonstrate that proper guidance of the two NC populations is crucial for a normal development of the anterior segment of the eye.