Bioenergetic Profiling of Zebrafish Embryonic Development

Abstract

Many debilitating conditions are linked to bioenergetic defects. Developing screens to probe the genetic and/or chemical basis for such links has proved intractable. Furthermore, there is a need for a physiologically relevant assay of bioenergetics in whole organisms, especially for early stages in life where perturbations could increase disease susceptibility with aging. Thus, we asked whether we could screen bioenergetics and mitochondrial function in the developing zebrafish embryo. We present a multiplexed method to assay bioenergetics in zebrafish embryos from the blastula period (3 hours post-fertilization, hpf) through to hatching (48 hpf). In proof of principle experiments, we measured respiration and acid extrusion of developing zebrafish embryos. We quantified respiratory coupling to various bioenergetic functions by using specific pharmacological inhibitors of bioenergetic pathways. We demonstrate that changes in the coupling to ATP turnover and proton leak are correlated with developmental stage. The multiwell format of this assay enables the user to screen for the effects of drugs and environmental agents on bioenergetics in the zebrafish embryo with high sensitivity and reproducibility.

Metabolic profile analysis of a single developing zebrafish embryo via monitoring of oxygen consumption rates within a microfluidic device

“…The final concentrations of the pharmacological inhibitors used in our experiments were prepared according to the data of Stackley et al 10 The final concentration of the oligomycin was 9.4 lM for all stages of embryos; 1.875 lM FCCP for 3-12 hpf and 2.5 lM FCCP for 24-48 hpf; and 6.25 lM sodium azide for 3-36 hpf and 1.25 lM sodium azide for 48 hpf. The pharmacological inhibitors, which were used at these final concentrations, were able to produce the maximum change in respiration without inducing death within the experimental timeframe.…”

“…The average OCR values of a single embryo at 3 hpf and 24 hpf were 20.23 and 63.45 pmol O 2 /min/embryo, which are close to the values of 13.5 and 60.2 pmol O 2 /min/embryo that were previously validated by utilizing the commercial microplate-based XF-24 analyzer. 10 B. Total basal respiration during the development of a single zebrafish embryo Figure 6 shows the total basal respiration (OCR, in pmol O 2 /min/embryo) during the development of a single zebrafish embryo for the blastula (3 hpf), gastrula (7 hpf), segmentation (12 hpf), segmentation/pharyngula transition (24 hpf), mid-pharyngula (36 hpf), and hatching (48 hpf) stages inside a microfluidic device.…”

“…The linear increase in total respiration measured by our proposed method is consistent with previous reports. 10,14 The increase in total respiration has been proven for the most part to correspond to the increase in mitochondrial quantity with embryonic age, as measured by the mitochondrial protein, COX IV (an index of mitochondrial content). 10 …”

“…8 is consistent with reports by other groups who used the commercial microplate-based extracellular flux (XF-24) analyzer to assess respiration and the coupling of respiration to mitochondrial function. 10 The mechanisms for the change in the mitochondrial and non-mitochondrial respiration correlated with zebrafish embryonic development still require intensive study in conjunction with other biochemical assays to provide additional information; determining these mechanisms could be outside the scope of this study. Our proposed platform demonstrates the feasibility for the first time of studying bioenergetic metabolism in vivo in a single developing zebrafish embryo inside a microfluidic device, providing a physiologically relevant view of the in vivo respiration and metabolic profile.…”

“…10 However, these measurements require the use of specialized 96-well microplates to perform the respiratory measurements; a capture screen is added on the top of each well to ensure that the embryos remain in the measurement chamber throughout the assay. A small chamber volume was temporarily created by lowering a piston-like probe into the well for the respiratory measurements.…”