Light-sheet fluorescence microscopy for the in vivo study of microtubule dynamics in the zebrafish embryo

Abstract

During its first hours of development, the zebrafish embryo presents a large microtubule array in the yolk region, essential for its development. Despite of its size and dynamic behavior, this network has been studied only in limited field of views or in fixed samples. We designed and implemented different strategies in Light Sheet Fluorescence microscopy for imaging the entire yolk microtubule (MT) network in vivo. These have allowed us to develop a novel image analysis from which we clearly observe a cyclical re-arrangement of the entire MT network in synchrony with blastoderm mitotic waves. These dynamics also affect a previously unreported microtubule array deep within the yolk, here described. These findings provide a new vision of the zebrafish yolk microtubules arrangement, and offers novel insights in the interaction between mitotic events and microtubules reorganization.

Multiple asters organize the yolk microtubule network during dclk2-GFP zebrafish epiboly

“…1 B) 12 , 13 . Additionally, we can also observe the recently reported third array of MT deep inside the yolk cell 10 .…”

“…We have primarily used dclk2-GFP embryos as a MT reporter transgenic line to study yolk MT organization. Thanks to our custom-made high throughput multi-view light-sheet imaging approach 10 , 28 , we have access, with high temporal (0.3–1 volume/min) and spatial (0.65–1.3 µm) resolutions, to the whole embryonic sphere throughout zebrafish early developmental stages. Compared to CLSM, LSFM provides high signal-to-noise ratio (SNR) images more efficiently, with reduced photo-bleaching and photo-toxicity 29 , 30 .…”

“…The Tg(ef1α:dclk-GFP) transgene contains two doublecortin domains (microtubule-binding domain) of zebrafish dclk1 fused to gfp, and under the control of the Xenopus ef1α promoter. In this work, we use it to image epiboly stages with LSFM, following a previous study in our lab by which the in toto observation of this specific transgenic line has allowed to underscore the dynamics of the MT network prior to epiboly stages 10 .…”

“…It is known that the zebrafish YSL and YCL contain MT networks that undergo various changes (MT density, MT length) over the different developmental stages [10][11][12][13][14] . Given its size and shape, the zebrafish yolk cell arises as an ideal model for studying how MT-based spatial organization scales with size, how it relates to geometrical constraints, and how its global architecture reshapes over time.…”

Dynamics of Drosophila endoderm specification

“…The MS2-MCP system has not been implemented in Ciona , but previous studies have successfully utilized fluorescently tagged proteins for similar aims ( 63 , 64 ). Zebrafish embryos can also be imaged with light-sheet microscopy ( 65 , 66 ), and MS2 reporters have previously been used within this developmental system ( 37 ). Nonetheless, difficulties may arise when considering higher-level vertebrates.…”