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Expansion Microscopy of Zebrafish for Neuroscience and Developmental Expansion microscopy of zebrafish for neuroscience PNAS PLUS and developmental biology studies Limor Freifelda, Iris Odstrcilb, Dominique Försterc, Alyson Ramirezb, James A. Gagnonb, Owen Randlettb, Emma K. Costad, Shoh Asanoa, Orhan T. Celikere, Ruixuan Gaoa,f, Daniel A. Martin-Alarcong, Paul Reginatog,h, Cortni Dicka, Linlin Chena,i, David Schoppikj,k,l, Florian Engertb, Herwig Baierc, and Edward S. Boydena,d,e,f,m,1 aMedia Lab, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139; bDepartment of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138; cDepartment Genes–Circuits–Behavior, Max Planck Institute of Neurobiology, Martinsried 82152, Germany; dDepartment of Brain and Cognitive Sciences, MIT, Cambridge, MA 02139; eDepartment of Electrical Engineering and Computer Science, MIT, Cambridge, MA 02139; fMcGovern Institute for Brain Research, MIT, Cambridge, MA 02139; gDepartment of Biological Engineering, MIT, Cambridge, MA 02139; hDepartment of Genetics, Harvard Medical School, Cambridge, MA 02138; iNeuroscience Program, Wellesley College, Wellesley, MA 02481; jDepartment of Otolaryngology, New York University School of Medicine, New York, NY 10016; kDepartment of Neuroscience and Physiology, New York University School of Medicine, New York, NY 10016; lNeuroscience Institute, New York University School of Medicine, New York NY 10016; and mCenter for Neurobiological Engineering, MIT, Cambridge, MA 02139 Edited by Lalita Ramakrishnan, University of Cambridge, Cambridge, United Kingdom, and approved October 25, 2017 (received for review April 17, 2017) Expansion microscopy (ExM) allows scalable imaging of preserved nections in the intact brain, using circuitry responsible for the 3D biological specimens with nanoscale resolution on fast vestibulo-ocular reflex (11–13) and the escape response (14) as diffraction-limited microscopes. Here, we explore the utility of ExM testbeds. Finally, ExM helped visualize the subsynaptic organization in the larval and embryonic zebrafish, an important model organism of neurotransmitter receptors, revealing the ring-like organization for the study of neuroscience and development. Regarding neurosci- of glycine receptors on the Mauthner (M) cell of the zebrafish (15, ence, we found that ExM enabled the tracing of fine processes of 16). Toward answering questions in development, we examined radial glia, which are not resolvable with diffraction-limited micros- nuclear architecture in gastrulating zebrafish embryos. In particular, copy. ExM further resolved putative synaptic connections, as well as we characterized nuclear envelope invaginations [previously de- molecular differences between densely packed synapses. Finally, ExM scribed in other cell types and species (17–27)],aswellasthe could resolve subsynaptic protein organization, such as ring-like configuration of nearby microtubules, and found that such struc- structures composed of glycine receptors. Regarding development, tures could be easily visualized via ExM across the zebrafish em- we used ExM to characterize the shapes of nuclear invaginations and bryo. The scalability of ExM allowed us to detect channels passing channels, and to visualize cytoskeletal proteins nearby. We detected through the nucleus at specific, and potentially rare, points in the nuclear invagination channels at late prophase and telophase, cycle of cell division, which may help illuminate the mechanics of potentially suggesting roles for such channels in cell division. Thus, ExM of the larval and embryonic zebrafish may enable systematic this process. ExM helped with the visualization of chromatin with studies of how molecular components are configured in multiple regard to molecular markers such as microtubules, which may help contexts of interest to neuroscience and developmental biology. unravel how the structural and molecular organization of cells leads to successful mitosis in embryogenesis and development. zebrafish | superresolution | microscopy | brain Methods Fish Maintenance and Care. All zebrafish (Danio rerio) larvae were raised in fish xpansion microscopy (ExM) enables 3D nanoscale resolution facility water at Harvard University according to protocols and procedures Eimaging of extended biological specimens by forming a dense, approved by the Harvard University/Faculty of Arts & Sciences Standing permeating, interconnected mesh of polyelectrolyte polymer Committee on the Use of Animals in Research and Teaching (Institutional throughout the specimen and then swelling the gel so as to move Animal Care and Use Committee), with the following exceptions: Larvae used key biomolecules or labels apart from each other (1). In our recently developed protein retention ExM (proExM) protocol, Significance an antibody-stained tissue is embedded in such a polyelectrolyte gel and proteins are covalently linked to the gel (2). The tissue is We explore the utility of expansion microscopy (ExM) in neu- then proteolytically digested to mechanically homogenize the roscience and developmental biology using the zebrafish specimen in a fashion that spares the antibody stain, followed by model. Regarding neuroscience studies, ExM enables the trac- addition of water to isotropically expand the gel-specimen ing of cellular processes in the zebrafish brain, as well as the composite. proExM linearly expands preserved cells and tissues imaging of synapses and their biomolecular content and or- ∼ by a factor of 4.5-fold, enabling a resolution improvement by ganization. Regarding development, ExM enables the resolving this magnitude on standard, diffraction-limited microscopes of nuclear compartments, particularly nuclear invaginations (e.g., for a ∼300-nm diffraction limit lens, a final effective res- and channels, and helps relate such cellular nanostructures to olution of ∼300/4.5 or ∼60–70 nm is obtained). Since proExM- proteins of the cytoskeleton during embryogenesis. processed tissues can be imaged on fast, diffraction-limited mi- croscopes, it enables scalable superresolution imaging, and thus Author contributions: L.F., I.O., D.F., A.R., J.A.G., O.R., D.A.M.-A., D.S., F.E., H.B., and E.S.B. may help open up the systematic exploration of how molecules designed research; L.F., I.O., D.F., A.R., J.A.G., O.R., E.K.C., O.T.C., R.G., D.A.M.-A., P.R., C.D., and L.C. performed research; L.F., D.F., S.A., and E.S.B. analyzed data; and L.F. and are configured throughout cells and tissues with nanoscale precision. NEUROSCIENCE E.S.B. wrote the paper. Here, we explore the application of proExM to zebrafish, a Conflict of interest statement: E.S.B. and R.G. are inventors on one or more patent appli- genetically tractable vertebrate that is transparent throughout cations related to expansion microscopy (ExM). E.S.B. is co-founder of a company, Expan- early life and, accordingly, has proven to be a useful model for sion Technologies, that aims to provide ExM kits and services to the community. neuroscience (3) and developmental biology (4). We explored This article is a PNAS Direct Submission. topics in both fields. Regarding neuroscience, we show that ExM This open access article is distributed under Creative Commons Attribution-NonCommercial- can enable the tracing of cellular processes too fine to trace in NoDerivatives License 4.0 (CC BY-NC-ND). diffraction-limited images [a common problem in neuroscience 1To whom correspondence should be addressed. Email: [email protected]. – (5, 6)], using radial glia of the tectum (7 10)asatestbed.ExM This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. also enabled the detection and molecular analysis of synaptic con- 1073/pnas.1706281114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1706281114 PNAS | Published online November 21, 2017 | E10799–E10808 Downloaded by guest on September 29, 2021 in Fig. 1 and SI Appendix,Fig.S1A, B,andD and one of two larvae used for A ’ Optic tectum the analysis shown in SI Appendix,Fig.S1C were raised in Danieau s medium at the Max Planck Institute of Neurobiology. These animal procedures conformed to the institutional guidelines of the Max Planck Society and the local gov- ernment (Regierung von Oberbayern). Experimental protocols were approved by Regierung von Oberbayern (55.2-1-54-2532-101-12 and 55.2-1-54-2532-31- pre-expansion post-expansion 2016). All larvae were raised on a standard 14-h light/10-h dark cycle at a temperature of 28 °C. B B’ Transgenic Fish Lines. The genotypes of the larvae and embryos used to RG2 RG1 generate each figure are detailed in SI Appendix, SI Methods. The transgenic RG2 fish lines that were crossed to produce these larvae and embryos were all RG1 previously described. All larval brain images are from 6-d postfertiliza- tion larvae, and all embryo images are from shield-stage (i.e., ∼6-h postfertilization) embryos. Immunohistochemistry. Immunohistochemistry was performed following standard, previously published procedures (28). The exact protocol, as well as a detailed list of antibodies used, is provided in SI Appendix, SI Methods. Expansion. Expansion was performed using the previously described proExM protocol (2) (SI Appendix, SI Methods). Imaging. Both pre- and postexpansion brains and embryos were imaged on an Andor spinning disk (CSU-X1 Yokogawa) confocal system with a 40×,1.15N.A. water immersion objective (Nikon), with the exception of some images in SI Appendix, Fig. S1: The first and third images in SI Appendix, Fig. S1A and all preexpansion images used to generate SI Appendix, Fig. S1C
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