Automated heart rate estimation in fish embryo

International audienceTransparent organisms such as fish embryos are being increasingly used for environmental toxicology studies. These studies require estimating a number of physiological parameters. These estimations may be diverse in nature and can be a challenge to automate. Among these, an example is the development of reliable and repeatable automated assays for the determination of heart rates. To achieve this, most existing method rely on cyclical luminance variations, since as the heart fills and empties, it become respectively brighter and darker. However, sometimes direct measurement of the heart rate may be difficult, depending on the age of the embryo, its actual transparency, and its aspect under the microscope. It may be easier to seek an indirect measurement. In this article, we estimate the heart function parameters, such as heart frequency, either from measuring the heart motion or from blood flow in arteries. This measurement is more complex from the image analysis point of view, but it is more precise, more physically meaningful and easier to use in practice and to automate than measuring illumination changes. It may also be more informative. We illustrate on medaka embryos

Quantifying function in the zebrafish embryonic heart: a study on the role of timed mechanical cues

2014 Summer.Congenital heart defects are a relatively common problem, yet the cause is unknown in the large majority of cases. A significant amount of past research has shown that there is a link between altered blood-induced mechanical stress and heart development. However, very little research has been done to examine the effect of altered loading timing. During embryonic development, the heart undergoes a drastic change in morphology from its original valveless tube structure to a complete multi-chambered pump with valves. Blood flow dynamics are consequently altered significantly as well. Given the changes occurring through this period, it is hypothesized that significant and persistent decreases in heart function occur when cardiac loading is altered during certain time windows of early development. The main objectives of this work were to (1) develop a methodology to quantify heart function in the embryonic zebrafish from high-speed bright field images, (2) develop a model for temporary and noninvasive alteration of cardiac loading, and (3) apply the methodology to normal and treated embryos to determine whether certain time windows of altered loading are more impactful than others. Results indicated that altered loading during the tube and early looping stages of development produce persistent changes in heart morphology along with accompanying decreases in cardiac function. Altered loading during late cardiac looping resulted in temporary changes in function which did not persist through the latest time point measured. This work has produced extensive tools for quantifying heart function from high speed images and presents a new model for altered cardiac loading in the zebrafish. Results support the hypothesis that the heart is more sensitive to altered loading during certain windows in development. This provides new insight into how congenital defects may develop and sets the stage for future experiments investigating the effects of altered loading on heart development

Quantitative analysis of the mechanical environment in the embryonic heart with respect to its relationship in cardiac development

Includes bibliographical references.2015 Fall.In order to understand the causes of congenital heart defects, which afflict at least 4 infants per 1,000 live births, research has implemented the use of animal models to study embryonic heart development. Zebrafish (Danio rerio) have become one of the more prominent of these animal models due to the fact that their heart morphology at the earliest stages of development is remarkably similar to humans, and because embryos lack pigmentation, rendering them transparent. This transparency allows for high-speed images of blood flow to be acquired in the developing heart so that the mechanotransductive relationship between the intracardiac flow environment and myocardial progenitor cell differentiation can be understood. One particular aspect of the flow environment, a cyclic retrograde flow at the junction of the forming atrium and ventricle, has been shown to be necessary for valve formation, though the mechanisms causing it to occur had previously been unknown. By comparing the results of two-dimensional spatiotemporal analysis applied to embryos both with normal retrograde flow and inhibited retrograde flow, this study shows that a particular range of pressures associated with the pumping mechanics of the heart as well as resistance due to systolic contractile closure must exist in order to maintain adequate retrograde flow to induce valve formation. The use of two-dimensional spatiotemporal analysis was sufficient to acquire these results, however when applied to analysis of other aspects of the intracardiac flow environment, this computational method is subject to critical limitations. Therefore, this study includes the development of methodology to integrate the results of spatiotemporal analysis on multiple focal planes bisecting the heart into a more accurate, three-dimensional result. The results of this study not only increase our understanding of the mechanics behind an important factor in embryonic development, but also enable future experiments pertaining to the measurement of embryonic intracardiac blood flow to be performed with increased certainty

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

Visualising apoptosis in live zebrafish using fluorescence lifetime imaging with optical projection tomography to map FRET biosensor activity in space and time

Fluorescence lifetime imaging (FLIM) combined with optical projection tomography (OPT) has the potential to map Förster resonant energy transfer (FRET) readouts in space and time in intact transparent or near transparent live organisms such as zebrafish larvae, thereby providing a means to visualise cell signalling processes in their physiological context. Here the first application of FLIM OPT to read out biological function in live transgenic zebrafish larvae using a genetically expressed FRET biosensor is reported. Apoptosis, or programmed cell death, is mapped in 3-D by imaging the activity of a FRET biosensor that is cleaved by Caspase 3, which is a key effector of apoptosis. Although apoptosis is a naturally occurring process during development, it can also be triggered in a variety of ways, including through gamma irradiation. FLIM OPT is shown here to enable apoptosis to be monitored over time, in live zebrafish larvae via changes in Caspase 3 activation following gamma irradiation at 24 hours post fertilisation. Significant apoptosis was observed at 3.5 hours post irradiation, predominantly in the head region

Adult and Developing Zebrafish as Suitable Models for Cardiac Electrophysiology and Pathology in Research and Industry

The electrophysiological behavior of the zebrafish heart is very similar to that of the human heart. In fact, most of the genes that codify the channels and regulatory proteins required for human cardiac function have their orthologs in the zebrafish. The high fecundity, small size, and easy handling make the zebrafish embryos/larvae an interesting candidate to perform whole animal experiments within a plate, offering a reliable and low-cost alternative to replace rodents and larger mammals for the study of cardiac physiology and pathology. The employment of zebrafish embryos/larvae has widened from basic science to industry, being of particular interest for pharmacology studies, since the zebrafish embryo/larva is able to recapitulate a complete and integrated view of cardiac physiology, missed in cell culture. As in the human heart, I-Kr is the dominant repolarizing current and it is functional as early as 48 h post fertilization. Finally, genome editing techniques such as CRISPR/Cas9 facilitate the humanization of zebrafish embryos/larvae. These techniques allow one to replace zebrafish genes by their human orthologs, making humanized zebrafish embryos/larvae the most promising in vitro model, since it allows the recreation of human-organ-like environment, which is especially necessary in cardiac studies due to the implication of dynamic factors, electrical communication, and the paracrine signals in cardiac functionThis work was supported by grants from the Gobierno Vasco PIBA2018-58 and GIC18/150. MH-V was supported by the Government of Extremadura (Grant No. TA18052

Noninvasive technique for measurement of heartbeat regularity in zebrafish (Danio rerio) embryos

<p>Abstract</p> <p>Background</p> <p>Zebrafish (<it>Danio rerio</it>), due to its optical accessibility and similarity to human, has emerged as model organism for cardiac research. Although various methods have been developed to assess cardiac functions in zebrafish embryos, there lacks a method to assess heartbeat regularity in blood vessels. Heartbeat regularity is an important parameter for cardiac function and is associated with cardiotoxicity in human being. Using stereomicroscope and digital video camera, we have developed a simple, noninvasive method to measure the heart rate and heartbeat regularity in peripheral blood vessels. Anesthetized embryos were mounted laterally in agarose on a slide and the caudal blood circulation of zebrafish embryo was video-recorded under stereomicroscope and the data was analyzed by custom-made software. The heart rate was determined by digital motion analysis and power spectral analysis through extraction of frequency characteristics of the cardiac rhythm. The heartbeat regularity, defined as the rhythmicity index, was determined by short-time Fourier Transform analysis.</p> <p>Results</p> <p>The heart rate measured by this noninvasive method in zebrafish embryos at 52 hour post-fertilization was similar to that determined by direct visual counting of ventricle beating (<it>p </it>> 0.05). In addition, the method was validated by a known cardiotoxic drug, terfenadine, which affects heartbeat regularity in humans and induces bradycardia and atrioventricular blockage in zebrafish. A significant decrease in heart rate was found by our method in treated embryos (<it>p </it>< 0.01). Moreover, there was a significant increase of the rhythmicity index (p < 0.01), which was supported by an increase in beat-to-beat interval variability (<it>p </it>< 0.01) of treated embryos as shown by Poincare plot.</p> <p>Conclusion</p> <p>The data support and validate this rapid, simple, noninvasive method, which includes video image analysis and frequency analysis. This method is capable of measuring the heart rate and heartbeat regularity simultaneously via the analysis of caudal blood flow in zebrafish embryos. With the advantages of rapid sample preparation procedures, automatic image analysis and data analysis, this method can potentially be applied to cardiotoxicity screening assay.</p

Development of Novel Transgenic Zebrafish Models and their Application to Studies on Environmental Oestrogens

Oestrogenic chemicals have become increasingly associated with health effects in wildlife populations and humans. Transgenic animal models have been developed to understand the mechanisms by which these oestrogenic chemicals alter hormonal signalling pathways and how these alterations can lead to chronic health effects. The use of highly informative transgenic animal models will also result in better use and potential reduction of intact animals used in animal testing in line with the principles of the 3Rs. In this thesis work, two novel oestrogen responsive transgenic zebrafish models have been generated to investigate the effects of oestrogenic chemicals, identify their tissue targets and better understand the temporal dynamics of these responses. Both models express the pigment-free ‘Casper’ (a mutant line lacking skin pigment) phenotype, which facilitate identification of responding target tissues in the whole fish in all fish life stages (embryos to adults). The oestrogen response element green fluorescent (ERE-GFP)-Casper model was generated by crossing an established ERE-GFP line with the skin pigment free Casper line. The model generated is highly sensitive to oestrogenic chemicals, detecting responses to environmentally relevant concentrations of EE2, bisphenol A (BPA), genistein and nonylphenol. Use of the ERE-GFP- Casper model shows chemical type and concentration dependence for green fluorescent protein (GFP) induction and both spatial and temporal responses for different environmental oestrogens tested. A semi-automated (ArrayScan) imaging and image analysis system was also developed to quantify whole body fluorescence responses for a range of different oestrogenic chemicals in the new transgenic zebrafish model. The zebrafish model developed provides a sensitive and highly integrative system for identifying oestrogenic chemicals, their target tissues and effect concentrations for exposures in real time and across different life stages. It thus has application for chemical screening to better direct health effects analysis of environmental oestrogens and for investigating the functional roles of oestrogens in vertebrates. The second model generated was an ERE-Kaede-Casper line developed via crossing of the ERE-GFP-Casper line and a UAS-Kaede line and screening subsequent generations for a desired genotype and homozygous expression of the transgenes. Kaede is a photoconvertible fluorescent protein that initially fluoresces green in colour and can be permanently converted to red fluorescence upon short exposure to UV light. The model has a silenced skin pigmentation and high sensitivity to oestrogenic chemicals comparable with the previously developed ERE-GFP-Casper model. Use of this model has identified windows of tissue-specific sensitivity to ethinyloestradiol (EE2) for exposure during early-life (0-48hpf) and illustrated that exposure to oestrogen (EE2) during early life (0-48hpf) can enhance responsiveness (sensitivity) to different environmental oestrogens (EE2, genistein and bisphenol A) for subsequent exposures during development. These findings illustrate the importance of oestrogen exposure history in effects assessments and they have wider implications for the possible adverse effects associated with oestrogen exposure.Biotechnology and Biological Sciences Research Council (BBSRC)AstraZenec