The circadian clock and the hypoxic signaling pathway play critical roles in physiological homeostasis as well as in tumorgenesis. Interactions between both pathways have repeatedly been reported for mammals during the last decade, the molecular basis, though, has not been identified so far. Expression levels of oxygen-regulated and circadian clock genes in zebrafish larvae (Danio rerio) and zebrafish cell lines were significantly altered under hypoxic conditions. Thus, long-term hypoxic incubation of larvae resulted in a dampening of the diurnal oscillation amplitude of the period1 gene expression starting only several hours after start of the hypoxic incubation. A significant decrease in the amplitude of the period1 circadian oscillation in response to hypoxia and in response to the hypoxic mimic CoCl2 was also observed using a zebrafish luciferase reporter cell line in constant darkness. In addition, activity measurements of zebrafish larvae using an infrared-sensitive camera demonstrated the loss of their usual circadian activity pattern under hypoxic conditions. To explore the functional basis of the observed cross-talk between both signaling pathways ChIP assays were performed. Increasing with the duration of hypoxia, a nearly 4-fold occupancy of hypoxia-inducible factor 1 (Hif-1α) at two specific E-box binding sites located in the period1 gene control region was shown, demonstrating therewith the transcriptional co-regulation of the core clock gene by the major transcription factor of the hypoxic pathway. On the other hand, circadian transgenic zebrafish cells, simulating a repressed or an overstimulated circadian clock, modified gene transcription levels of oxygen-regulated genes such as erythropoietin and vascular endothelial growth factor 165 and altered the hypoxia-induced increase in Hif-1α protein concentration. In addition, the amount of Hif-1α protein accumulated during the hypoxic response was shown to depend on the time of the day, with one maximum during the light phase and a second one during the dark phase. The direct binding of Hif-1α to the period1 gene control region provides a mechanistic explanation for the repeatedly observed interaction between hypoxia and the circadian clock. The cross-talk between both major signaling pathways was shown for the first time to be bidirectional and may provide the advantage of orchestrating a broad range of genes and metabolic pathways to cope with altered oxygen availabilities.

Mutations in the Tulp1 gene cause severe, early-onset retinitis pigmentosa (RP14) in humans. In the retina, Tulp1 is mainly expressed in photoreceptors that use ribbon synapses to communicate with the inner retina. In the present study, we demonstrate that Tulp1 is highly enriched in the periactive zone of photoreceptor presynaptic terminals where Tulp1 colocalizes with major endocytic proteins close to the synaptic ribbon. Analyses of Tulp1 knock-out mice demonstrate that Tulp1 is essential to keep endocytic proteins enriched at the periactive zone and to maintain high levels of endocytic activity close to the synaptic ribbon. Moreover, we have discovered a novel interaction between Tulp1 and the synaptic ribbon protein RIBEYE, which is important to maintain synaptic ribbon integrity. The current findings suggest a new model for Tulp1-mediated localization of the endocytic machinery at the periactive zone of ribbon synapses and offer a new rationale and mechanism for vision loss associated with genetic defects in Tulp1.

In recent years, natural and anthropogenic factors have increased aquatic hypoxia the world over. In most organisms, the cellular response to hypoxia is mediated by the master regulator hypoxia-inducible factor-1 (HIF-1). HIF-1 also plays a critical role in the normal development of the cardiovascular system of vertebrates. We tested the hypothesis that hypoxia exposures which resulted in HIF-1 induction during embryogenesis would be associated with enhanced hypoxia tolerance in subsequent developmental stages. We exposed zebrafish (Danio rerio) embryos to just 4 h of severe hypoxia or total anoxia at 18, 24 and 36 h post-fertilization (hpf). Of these, exposure to hypoxia at 24 and 36 hpf as well as anoxia at 36 hpf activated the HIF-1 cellular pathway. Zebrafish embryos that acutely upregulated the HIF-1 pathway had an increased hypoxia tolerance as larvae. The critical window for hypoxia sensitivity and HIF-1 signalling was 24 hpf. Adult male fish had a lower critical oxygen tension (P crit ) compared with females. Early induction of HIF-1 correlated directly with an increased proportion of males in the population. We conclude that mounting a HIF-1 response during embryogenesis is associated with long-term impacts on the phenotype of later stages which could influence both individual hypoxia tolerance and population dynamics.

Munc119 (also denoted as RG4) is a mammalian ortholog of the Caenorhabditis elegans protein unc119 and is essential for vision and synaptic transmission at photoreceptor ribbon synapses by unknown molecular mechanisms. Munc119/RG4 is related to the prenyl-binding protein PrBP/␦ and expressed at high levels in photoreceptor ribbon synapses. Synaptic ribbons are presynaptic specializations in the active zone of these tonically active synapses and contain RIBEYE as a unique and major component. In the present study, we identified Munc119 as a RIBEYE-interacting protein at photoreceptor ribbon synapses using five independent approaches. The PrBP/␦ homology domain of Munc119 is essential for the interaction with the NADH binding region of RIBEYE(B) domain. But RIBEYEMunc119 interaction does not depend on NADH binding. A RIBEYE point mutant (RE(B)E844Q) that no longer interacted with Munc119 still bound NADH, arguing that binding of Munc119 and NADH to RIBEYE are independent from each other. Our data indicate that Munc119 is a synaptic ribbon-associated component. We show that Munc119 can be recruited to synaptic ribbons via its interaction with RIBEYE. Our data suggest that the RIBEYE-Munc119 interaction is essential for synaptic transmission at the photoreceptor ribbon synapse.Munc119 (also denoted as RG4, Ref.

Exercise as well as hypoxia cause an increase in angiogenesis, changes in mitochondrial density and alterations in metabolism, but it is still under debate whether the hypoxia inducible factor (HIF) is active during both situations. In this study gene expression analysis of zebrafish larvae that were raised under normoxic, hypoxic, or training conditions were compared, using microarray analysis, quantitative real-time PCR and protein data. Although HIF expression is posttranslationally regulated, mRNA expression levels of all three isoforms (HIF-1α, HIF-2α, and HIF-3α) differed in each of the experimental groups, but the changes observed in hypoxic animals were much smaller than in trained larvae. Prominent changes were seen for Hif-2α expression, which significantly increased after the first day of exercise and then decreased down to values significantly below control values. HIF-3α mRNA expression in turn increased significantly, and at the end of the training period (9-15 days postfertilization) it was elevated three times. At the protein level a transient increase in HIF-1α was observed in hypoxic larvae, whereas in the exercise group the amount of HIF-1α protein even decreased below the level of control animals. The analyzed transcriptome was more affected in hypoxic zebrafish larvae, and hardly any genes were similarly altered by both treatments. These results clearly showed that HIF proteins played different roles in trained and hypoxic zebrafish larvae and that the exercise-induced transition to a more aerobic phenotype was not achieved by persistent activation of the hypoxic signaling pathway.