59 LIPID DYNAMICS IN THE EARLY EMBRYONIC DEVELOPMENT OF ZEBRAFISH BY DESORPTION ELECTROSPRAY IONIZATION MASS SPECTROMETRY IMAGING AND NANOELECTROSPRAY–MASS SPECTROMETRY

Abstract

This study introduces a combination of mass spectrometry-based analytical approaches with little or no sample preparation for the study of zebrafish (Danio rerio) lipid metabolism during early development (0 through 96 h post-fertilization, hpf). Desorption electrospray ionization mass spectrometry (DESI-MS) imaging and nanoelectrospray (nESI) MS were used. Embryos (N = 107) were placed onto filter paper squares and analysed by DESI-MS imaging. Embryos were arranged in rows with each one corresponding to a different developmental time, so that each DESI-MS image contained arrays of samples at different hpf. After completion of DESI-MS acquisition, the arrays were cut into strips of paper, isolating each embryo, which was subsequently inserted into a nESI tip and analysed. All experiments were performed using a linear ion trap mass spectrometer. DESI-MS images were acquired in the positive ion mode using acetonitrile doped with 10 ppm AgNO3, which allowed for the detection of cytosolic neutral lipids, such as cholesteryl esters (CE), diacylglicerols (DAG), triacylglycerols (TAG), squalene, and ubiquinone. For the nESI, experiments were run in full mass scan and MS/MS, in both negative and positive ion mode, using a mixture of dimethylformamide and acetonitrile to detect membrane phospholipids (e.g. phosphatidylcholines and phosphatidylglycerols). Principal component analysis was used to explore DESI-MS images and nESI data in an unsupervised fashion. The combination of DESI-MS imaging, providing chemical and spatial location, and nanoESI, providing a broader and more extensive structural information, showed that the lipid content changes dramatically over the first days of development. Accumulation of DAG and TAG, which are usually concentrated in the embryos yolk sac as an important storage for early development, were observed between 0 and 48 hpf. Embryos at the intermediate phase of development (24 hpf) were distributed between those of 0 and 48 hpf, reflecting the dynamics associated with development and might be correlated with TAG consumption and de novo synthesis. After 72 and 96 hpf, samples differed from prior developmental stages by the content of squalene, ubiquinone, and CE 22 : 5 (overexpressed), while the TAG content decreased. Further decrease of TAGs and ubiquinone occurred between 72 and 96 hpf. These observations indicated that zebrafish embryos rely entirely on the yolk sac for the nutrients needed to sustain growth and survival during the first four days of development. Yolk lipids are the likely source of TAGs, as well as cholesterol, a required component of cell membranes and a precursor for bile acids. The abundance of ubiquinone can be tied to zebrafish embryo growth, differentiation and organogenesis, and activation of mitochondria.