Marcus P. S. Dekens

Background: Recent epidemics of influenza, AIDS, SARS, Ebola, Zika, and COVID-19 highlight the persistent threat of RNA viruses to public health. COVID-19 exposed major gaps in monitoring capabilities, leaving lockdowns as the only means to reduce transmission. Moreover, their effectiveness was limited by economic pressures that forced early relaxation. Methods: To strengthen monitoring capabilities, we designed a scalable workflow for specimen collection, analysis, and reporting. Rapid implementation was achieved by repurposing non medical, nonprofit, laboratory infrastructure. We analyzed the variation in 4,295,664 viral genomes collected from patients worldwide using software developed inhouse. qPCR assays targeting stable regions of the pathogen’s genome were developed and then validated in a clinical performance study. Between September 2020 and December 2021, 476,502 specimens from local staff and caregivers were analyzed, 177,756 of which were processed in batches. Results: The effectiveness of this response model is demonstrated by the significant reduction in incidence ( P =0.007, d =4.54) among 2,475 systematically monitored caregivers compared with the general population. Performance validation of the assays showed high analytical sensitivity, enabling scaling up while detecting SARS-CoV-2 below the infectious viral load. Frequent testing with same-day notification was associated with effective control of viral transmission, comparing favorably with lockdowns. The high inclusive specificity (>99.99%) of the diagnostic panel further minimized outbreak risk. Interpretation: Timely identification and isolation of cases is essential to protect health systems, inform policy decisions, and support economic stability. Infectious diseases with pre- or asymptomatic transmission necessitate molecular diagnostic testing. Effective deployment requires dedicated workflows that integrate supply and information systems, combining high-throughput with fast, affordable results. This after-action report presents an operationally grounded framework for responding to emerging infectious diseases.

Mammalian and fish pineals play a key role in adapting behaviour to the ambient light conditions through the release of melatonin. In mice, light inhibits nocturnal locomotor activity via the non‐visual photoreceptor Melanopsin. In contrast to the extensively studied function of Melanopsin in the indirect regulation of the rodent pineal, its role in the intrinsically photosensitive zebrafish pineal has not been elucidated. Therefore, it is not evident if the light signalling mechanism is conserved between distant vertebrates, and how Melanopsin could affect diurnal behaviour. A double knockout of melanopsins ( opn4.1 ‐ opn4xb ) was generated in the diurnal zebrafish, which manifests attenuated locomotor activity during the wake state. Transcriptome sequencing gave insight into pathways downstream of Melanopsin, implying that sustained repression of the melatonin pathway is required to elevate locomotor activity during the diurnal wake state. Moreover, we show that light induces locomotor activity during the diurnal wake state in an intensity‐dependent manner. These observations suggest a common Melanopsin‐driven mechanism between zebrafish and mammals, while the diurnal and nocturnal chronotypes are inversely regulated downstream of melatonin.

A tailored protocol combines pigment removal with clearing, facilitating labeling and whole-body imaging across phylogeny.

The acquisition of reliable data strongly depends on experimental design. When studying the effects of light on processes such as behaviour and physiology it is crucial to maintain all environmental conditions constant apart from the one under study. Furthermore, the precise values of the environmental factors applied during the experiment should be known. Although seemingly obvious, these conditions are often not met when the effects of light are being studied. Here, we document and discuss the wavelengths and light intensities of natural and artificial light sources. We present standardised experimental protocols together with building plans of a custom made instrument designed to accurately control light and temperature for experiments using fresh water or marine species. Infrared light is commonly used for recording behaviour and in electrophysiological experiments although the properties of fish photoreceptors potentially allow detection into the far red. As an example of our experimental procedure we have applied our protocol and instrument to specifically test the impact of infrared light (840 nm) on the zebrafish circadian clock, which controls many aspects of behaviour, physiology and metabolism. We demonstrate that infrared light does not influence the zebrafish circadian clock. Our results help to provide a solid framework for the future study of light dependent processes in aquatic organisms.

Numerous responses are triggered by light in the cell. How the light signal is detected and transduced into a cellular response is still an enigma. Each zebrafish cell has the capacity to directly detect light, making this organism particularly suitable for the study of light dependent transcription. To gain insight into the light signalling mechanism we identified genes that are activated by light exposure at an early embryonic stage, when specialised light sensing organs have not yet formed. We screened over 14,900 genes using micro-array GeneChips, and identified 19 light-induced genes that function primarily in light signalling, stress response, and DNA repair. Here we reveal that PAR Response Elements are present in all promoters of the light-induced genes, and demonstrate a pivotal role for the PAR bZip transcription factor Thyrotroph embryonic factor (Tef) in regulating the majority of light-induced genes. We show that tefβ transcription is directly regulated by light while transcription of tefα is under circadian clock control at later stages of development. These data leads us to propose their involvement in light-induced UV tolerance in the zebrafish embryo.

On the first day of development a circadian clock becomes functional in the zebrafish embryo. How this oscillator is set in motion remains unclear. We demonstrate that zygotic period1 transcription begins independent of light exposure. Pooled embryos maintained in darkness and under constant temperature show elevated non‐oscillating levels of period1 expression. Consequently, there is no maternal effect or developmental event that sets the phase of the circadian clock. Analysis of period1 transcription, at the cellular level in the absence of environmental stimuli, reveals oscillations in cells that are asynchronous within the embryo. Demonstrating an autonomous onset to rhythmic period1 expression. Transcription of clock1 and bmal1 is rhythmic in the adult, but constant during development in light‐entrained embryos. Transient expression of dominant‐negative ΔCLOCK blocks period1 transcription, thus showing that endogenous CLOCK is essential for the transcriptional regulation of period1 in the embryo. We demonstrate a default mechanism in the embryo that initiates the autonomous onset of the circadian clock. This embryonic clock is differentially regulated from that in the adult, the transition coinciding with the appearance of several clock output processes.

Large-scale mutagenesis screens in the zebrafish employing the mutagen ENU have isolated several hundred mutant loci that represent putative developmental control genes. In order to realize the potential of such screens, systematic genetic mapping of the mutations is necessary. Here we report on a large-scale effort to map the mutations generated in mutagenesis screening at the Max Planck Institute for Developmental Biology by genome scanning with microsatellite markers. We have selected a set of microsatellite markers and developed methods and scoring criteria suitable for efficient, high-throughput genome scanning. We have used these methods to successfully obtain a rough map position for 319 mutant loci from the Tübingen I mutagenesis screen and subsequent screening of the mutant collection. For 277 of these the corresponding gene is not yet identified. Mapping was successful for 80 % of the tested loci. By comparing 21 mutation and gene positions of cloned mutations we have validated the correctness of our linkage group assignments and estimated the standard error of our map positions to be approximately 6 cM. By obtaining rough map positions for over 300 zebrafish loci with developmental phenotypes, we have generated a dataset that will be useful not only for cloning of the affected genes, but also to suggest allelism of mutations with similar phenotypes that will be identified in future screens. Furthermore this work validates the usefulness of our methodology for rapid, systematic and inexpensive microsatellite mapping of zebrafish mutations.

Zebrafish are typically used as a model system to study various aspects of developmental biology, largely as a consequence of their ex vivo development, high degree of transparency, and, of course, ability to perform forward genetic mutant screens. More recently, zebrafish have been developed as a model system with which to study circadian clocks. Cell lines generated from early‐stage zebrafish embryos contain clocks that are directly light‐responsive. We describe recent experiments using single‐cell luminescent imaging approaches to study clock function in this novel cell line system. Furthermore, studies examining the process of entrainment to light pulses within this cell population are described in this review, as are experiments examining light‐responsiveness of early‐stage zebrafish embryos.

In animal species, early developmental processes are driven by maternally derived factors. Here, we describe a forward genetics approach to identify recessive mutations in genes encoding such maternal factors in the zebrafish. We used a gynogenesis‐based approach to identify 14 recessive maternal‐effect mutations. Homozygosity for these mutations in adult females leads to the inviability of their offspring. Confocal microscopy of embryos labeled with a DNA dye and a membrane marker allowed us to further analyze mutant embryos for defects in nuclear and cellular divisions. The mutations result in a range of defects in early developmental processes, including egg activation, early nuclear events, mitosis, cytokinesis, axial patterning, and gastrulation. Our effort constitutes a systematic attempt to identify maternal‐effect genes in a vertebrate species. The sample of mutations that we have identified reflects the diversity of maternally driven functions in early development and underscores the importance of maternal factors in this process. Developmental Dynamics 231:324–335, 2004.