In vivo analysis reveals a switch in metabolism and oxidative state of zebrafish eyes during development

Abstract

Purpose : Metabolic and oxidative states fluctuate during normal organogenesis and tissue maturation, disruption of which can lead to disease. However, methods for observing these fluctuations in vivo have been lacking. We employed Fluorescence Lifetime Imaging Microscopy (FLIM) using non-invasive, 2-photon excitation to study intrinsic oxidative versus glycolytic metabolic and oxidative states in the eyes of living zebrafish (Danio rerio) embryos. At 2 dpf the developing lens is a ball of secondary fiber cells surrounded by the lens epithelium. At 3 dpf lens nuclear fiber cells have compacted and matured, lost their organelles, and all 5 major retinal cell types are formed.

Methods : Zebrafish “crystal” fish, which are triple mutants for nacre^w2/w2;alb^b4/b4 and roy^a9/a9, lack melanin, melanophores and iridophores, resulting in a transparent retinal pigment epithelium that allows optical access for in vivo imaging. Embryos were anesthetized, mounted in agarose and eyes were imaged using FLIM with 740 nm excitation. Optical slices of the eye were collected along the optical axis and data was analyzed using the phasor plot approach. To assess the free-to-bound NADH ratio (indicating metabolic state), and existence of long lifetime species (LLS, which indicates oxidative stress) from lens and retina we used a three components analysis. Masking enabled signal isolation from each anatomical region.

Results : An increase in free NADH and decrease in the oxidative state of oleic acid (decreased LLS ratio), was observed at 3 dpf compared to 2 dpf in both the lens and retina. Structural changes on high resolution imaging mirror published histology.

Conclusions : The shift from primarily oxidative phosphorylation to glycolytic metabolism in the lens likely reflects the degradation of mitochondria in mature lens fiber cells, which cannot undergo oxidative phosphorylation. A shift to glycolytic metabolism in the retina may reflect the high metabolic demand of newly formed photoreceptors at 3 dpf. The decrease in oxidation at 3 dpf compared to 2 dpf is consistent with previously published HPLC analysis of whole zebrafish embryos at these stages. This is the first non-invasive analysis of live zebrafish eyes revealing dramatic changes in metabolism and oxidative states during key developmental stages.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.