A Whole Brain Staining, Embedding, and Clearing Pipeline for Adult Zebrafish to Visualize Cell Proliferation and Morphology in 3-Dimensions.

Benjamin W Lindsey,Alon M Douek,Felix Loosli,Jan Kaslin

doi:10.3389/fnins.2017.00750

Abstract

The field of macro-imaging has grown considerably with the appearance of innovative clearing methods and confocal microscopes with lasers capable of penetrating increasing tissue depths. The ability to visualize and model the growth of whole organs as they develop from birth, or with manipulation, disease or injury, provides new ways of thinking about development, tissue-wide signaling, and cell-to-cell interactions. The zebrafish (Danio rerio) has ascended from a predominantly developmental model to a leading adult model of tissue regeneration. The unmatched neurogenic and regenerative capacity of the mature central nervous system, in particular, has received much attention, however tools to interrogate the adult brain are sparse. At present there exists no straightforward methods of visualizing changes in the whole adult brain in 3-dimensions (3-D) to examine systemic patterns of cell proliferation or cell populations of interest under physiological, injury, or diseased conditions. The method presented here is the first of its kind to offer an efficient step-by-step pipeline from intraperitoneal injections of the proliferative marker, 5-ethynyl-2′-deoxyuridine (EdU), to whole brain labeling, to a final embedded and cleared brain sample suitable for 3-D imaging using optical projection tomography (OPT). Moreover, this method allows potential for imaging GFP-reporter lines and cell-specific antibodies in the presence or absence of EdU. The small size of the adult zebrafish brain, the highly consistent degree of EdU labeling, and the use of basic clearing agents, benzyl benzoate, and benzyl alcohol, makes this method highly tractable for most laboratories interested in understanding the vertebrate central nervous system in health and disease. Post-processing of OPT-imaged adult zebrafish brains injected with EdU illustrate that proliferative patterns in EdU can readily be observed and analyzed using IMARIS and/or FIJI/IMAGEJ software. This protocol will be a valuable tool to unlock new ways of understanding systemic patterns in cell proliferation in the healthy and injured brain, brain-wide cellular interactions, stem cell niche development, and changes in brain morphology.

Highlights

How cells in the developing or adult brain are organized and behave following injury or disease remains a fascinating, yet still poorly understood, question
Histogram Analysis To examine changes in cell proliferation across the A-P neuro-axis of the adult zebrafish brain under homeostasis and following lesion, we developed an analysis method allowing us to plot the histogram of EdU intensity (Figure 4A). 3D reconstructed, EdU-stained brains were virtually sectioned through the horizontal plane, and a final maximum projection obtained
The specificity of EdU labeling in the dorsal forebrain adjacent the ventricle in virtual cross-sections from our Optical Projection Tomography (OPT) pipeline are consistent with immunolabeling using the common proliferative marker, Proliferating Cell Nuclear Antigen (PCNA) in this same domain

Summary

Introduction

How cells in the developing or adult brain are organized and behave following injury or disease remains a fascinating, yet still poorly understood, question. The value of macro-imaging has been demonstrated across a range of tissues, including embryos (Sharpe et al, 2002; Sharpe, 2003), heart (Kolesová et al, 2016; Aguilar-Sanchez et al, 2017), kidney (Short et al, 2010; Combes et al, 2014; Short and Smyth, 2016, 2017), lymph node (Song et al, 2015), mammary glands (Lloyd-Lewis et al, 2016), and brain (Gleave et al, 2013; Ode and Ueda, 2015), leading to new insight into the cellular behavior of organs under diverse conditions This progress has been facilitated by the power of multiphoton imaging, newer confocal microscopes with lasers having increasingly better z-axis penetration, the development of light-sheet microscopes, and tomographic techniques such as Optical Projection Tomography (Sharpe et al, 2002; Keller et al, 2010; Parra et al, 2012; Kromm et al, 2016; McGowan and Bidwell, 2016; Susaki and Ueda, 2016; Whitehead et al, 2017). Whole organ imaging of thick tissue of ∼1 mm or greater introduce a number of challenges that must be overcome compared to antibody labeling and confocal imaging of sectioned tissue at the micron scale

Methods

Results

Conclusion