Expansion microscopy of zebrafish for neuroscience and developmental biology studies

Abstract

Expansion microscopy (ExM) allows scalable imaging of preserved 3D biological specimens with nanoscale resolution on fast diffraction-limited microscopes. Here, we explore the utility of ExM in the larval and embryonic zebrafish, an important model organism for the study of neuroscience and development. Regarding neuroscience, we found that ExM enabled the tracing of fine processes of radial glia, which are not resolvable with diffraction-limited microscopy. ExM further resolved putative synaptic connections, as well as molecular differences between densely packed synapses. Finally, ExM could resolve subsynaptic protein organization, such as ring-like structures composed of glycine receptors. Regarding development, we used ExM to characterize the shapes of nuclear invaginations and channels, and to visualize cytoskeletal proteins nearby. We detected nuclear invagination channels at late prophase and telophase, potentially suggesting roles for such channels in cell division. Thus, ExM of the larval and embryonic zebrafish may enable systematic studies of how molecular components are configured in multiple contexts of interest to neuroscience and developmental biology.

Keywords: brain; microscopy; superresolution; zebrafish.

Fig. 1.

ExM helps resolve the morphology of fine cellular processes. (A) Schematic of the larval zebrafish brain showing the imaged area (red rectangle) within the left optic tectum. (B and B′) Maximum intensity projections of part of the tectum (highlighted in red in A) of a 6-d postfertilization larval zebrafish sparsely expressing membrane-bound EGFP and stained for GFP preexpansion (B) and postexpansion (B′), showing radial glial cells (two of which are labeled RG1 and RG2) and projection fiber bundles (arrowheads). (C and C′) Single confocal slices show projections of cell RG1, preexpansion (C) and postexpansion (C′). Endfeet processes of this cell wrap around the cell body of a superficial interneuron [SIN (113); arrow]. (D and D′) A bundle of extratectal fibers preexpansion (D) and postexpansion (D′) from the regions highlighted by arrowheads in B and B′, respectively, is shown. (E and E′) Intensity plots along the orange line in D and D′, respectively. AU, arbitrary units. [Scale bars: B, 10 μm; B′, 10 μm (physical size postexpansion, 35 μm); C and D, 5 μm; C′ and D′, 5 μm (17.5 μm).]

Fig. 2.

ExM analysis of synaptic connections. (A) Schematic of larval zebrafish brain showing nIII and nIV nuclei, labeled by Tg(isl1:GFP)rw0 (GFP, yellow) and neural projections labeled by Tg(−6.7FRhcrtR:Gal4VP16);Tg(UAS:Kaede) (Kaede, magenta). The rectangular area is imaged in B. (B) Maximal intensity projection of an ∼33-μm-thick volume corresponding to the rectangular area shown in A. The fish is 6 d postfertilization (dpf), and is stained with anti-GFP (yellow), anti-Kaede (magenta), and anti–pan-MAGUK (not shown). (C) GFP-labeled cells (yellow) and Kaede-labeled projections (magenta) in the nIII nucleus. (C, I–IV and I′–IV′) Two nearby planes (one in each row) from an expanded 6-dpf brain stained with anti-GFP (yellow), anti-Kaede (magenta), and anti-synaptotagmin2b (cyan). Arrows point to Kaede-expressing and synaptotagmin2b-stained varicosities and terminals next to GFP-labeled neuropil (IV) and cell bodies (IV′). Arrowheads point to a cluster of synaptotagmin2b, unlabeled by Kaede, next to a GFP-labeled cell (IV) and a Kaede-labeled synaptotagmin2b-stained varicosity next to a GFP-negative cell (IV′). (C, V–VIII) Single plane from a brain stained with anti–pan-MAGUK (cyan). Arrows point to Kaede-labeled varicosities and terminals next to GFP-labeled cells and neuropil, exhibiting colocalized MAGUK puncta. Arrowheads point to a MAGUK punctum on a GFP-negative cell opposed to a Kaede-labeled terminal (Top arrowhead) and to MAGUK puncta on GFP-labeled cell bodies and neuropil in the absence of nearby Kaede-labeled projections (Bottom two arrowheads). [Scale bars: B, 10 μm (38 μm); C, I–IV and I′–IV′, 5 μm (23 μm); C, V–VIII, 5 μm (19 μm).]

Fig. 3.

Expansion enables the resolving of synaptic heterogeneity and structure in intrasynaptic protein distributions. (A) Schematic of a larval zebrafish brain showing the M cells (blue) and spiral fiber neurons (magenta). The rectangle illustrates the region focused in on in B–D, consisting of the axon cap and a part of the M cell body. (B) Preexpansion images of the axon-cap area showing spiral fiber neurons (magenta) wrapping around the M cell axon initial segment (the unlabeled “tube” passing through these fibers, better visualized as a black stripe in C), as well as synaptotagmin2b (Top, cyan) and glycine receptors (Bottom, cyan). (C) Same as in B, but postexpansion. (Note: The synaptotagmin2b axon cap shown (Top) is not from the same brain as in B, Top.) (Top Left and Right) Arrows point to a Kaede-labeled varicosity bearing synaptotagmin2b at a low density. (Top Left and Right) Arrowheads point to a Kaede-negative varicosity bearing dense synaptotagmin2b staining. (Center) Arrowheads point to varicosities in spiral fiber neuron projections forming the M cell axon cap. (D) Maximal intensity projection of the M cell body and axon initial segment area showing the distribution of glycine receptors (cyan) preexpansion (Left) vs. postexpansion (Right). (Note: This is the same axon cap as shown in B and C, Bottom.) Boxes highlight seven examples of ring-shaped clusters zoomed in on in E. (E) Seven examples of ring-shaped clusters of various sizes present on the M cell body (1–6), and axon (7). [Scale bars: B, Top and Bottom, 5 μm; C, Top, 5 μm (23 μm); C, Bottom, 5 μm (20 μm); D, Left, 5 μm; D, Right, 5 μm (20 μm); E, 1 μm (4 μm).]

Fig. 4.

ExM examination of intranuclear invaginations. In all panels in this figure, in Figs. 5 and 6, and in Movies S5–S8: blue, anti-lamin B; green, histone 2B [EGFP fused to histone 2B in Tg(actb2:h2b-egfp/actb2:mem-mCherry2) and then stained with anti-GFP]; red, anti–α-tubulin (microtubules). All nuclei in this figure are from the same shield-stage embryo, either preexpansion (A) or postexpansion (B). (A, I–IV) Plane within a nucleus from this embryo. (A, IV) Arrowhead points to a centrosome positioned at the exit point of an indentation ending within the nucleus. (A, V) Maximal intensity projection of the invagination-containing area of this nucleus. (B, I–III) Three planes within a nucleus, postexpansion. (B, IV–VI) Maximal intensity projection of the invagination-containing area of this nucleus (shown is the same projection in all three panels, with stains as defined above). [Scale bars: A, 5 μm; B, 5 μm (20.5 μm).]

Fig. 5.

ExM reveals intranuclear channels in late-prophase and telophase nuclei. (A) Nucleus at late prophase, postexpansion. (A, I–III) Three planes from the nucleus. (A, I) Arrowhead points to a centrosome at one opening of an intranuclear channel. (A, III) Arrowhead points to a centrosome at the other opening of the intranuclear channel. (A, IV–VI) Maximal intensity projection of the intranuclear channel-containing area, showing that the channel runs between the two centrosomes located at opposing edges of this nucleus. (B) Nucleus at late prophase, preexpansion, containing two centrosomes at opposite ends of a lamin B-stained channel. (B, I) Representative plane from the nucleus. (B, II–IV) Maximal intensity projection of the invagination-containing region of the nucleus. (C) Structure of a late-telophase nucleus, postexpansion. (Top) Schematic of the structure observed in 10 late-telophase nuclei (eight more nuclei are shown in SI Appendix, Fig. S12). Chromatin (green) is unpacked and fills the daughter nuclei that are surrounded by continuous lamin B staining (blue). Two microtubule bundles (red) diverge from their convergence point at the midbody toward each daughter nucleus and disperse when they approach the nucleus. Unbundled microtubules pass through a channel through each daughter nucleus to connect with the centrosome at the other end. Black boxes outline the areas indicated in I–IV. (C, I) Maximal intensity projection of the invagination-containing area in one of the two daughter nuclei, showing a channel traversing through this nucleus. The arrowhead points to a centrosome at the channel opening. (C, II–IV) Different planes showing the second daughter nucleus. (C, II) Arrow points to a dip in the intensity of microtubule staining at the midbody. (C, IV) Arrowhead points to a centrosome at the channel opening within the second daughter nucleus. [Scale bars: A, 5 μm (19 μm); B, 5 μm; C, 5 μm (20.5 μm).]

Fig. 6.

ExM and the analysis of mitotic nuclei. (A) Mitotic nuclei, postexpansion. (A, I–I′′) Nucleus at the transition between prophase and prometaphase. (A, I) Lamin B staining. (A, I′) Microtubules. Arrowheads point to three short microtubules emerging from one of the centrosomes. (A, I′′) Overlay. (A, II–II′′′) Nucleus at prometaphase. (A, II) Lamin B staining. (A, II′) Maximal intensity projection of the microtubules in this nucleus. (A, II′′ and II′′′) Two distinct planes in this nucleus. (A, II′′) Arrowheads point to microtubules at the boundaries of this nucleus. (A, II′′′) Arrowhead points to lamin B staining next to a microtubule protruding into this nucleus. (A, III and III′) Nucleus at late prometaphase. (A, III) Maximal intensity projection of microtubules in this nucleus. (A, III′) Plane from this nucleus. Arrowheads point to discontinuous patches of lamin B surrounding the nucleus. (A, IV) Plane from a nucleus at metaphase. (A, V and V′) Nucleus at early anaphase. (A, V) Maximal intensity projection of microtubules in this nucleus. (A, V′) Plane from this nucleus. (A, VI) Plane from a nucleus at anaphase. Arrowheads point to fine microtubules located between chromosomes. (A, VII) Plane from a nucleus at late anaphase. Arrowheads point to lamin B patches at the boundary of nuclear chromatin. (A, VIII) Plane from a nucleus at telophase. (A, IX) Plane from a nucleus at late telophase. (B) Mitotic nuclei, preexpansion. (B, I–I′′) Plane from a nucleus at late prometaphase. (B, I) Lamin B staining. (B, I′) Microtubules. (B, I′′) Overlay. (B, II–II′′) Plane from a nucleus at metaphase. (B, II) Lamin B staining. (B, II′) Microtubules. (B, II′′) Overlay. (B, III) Plane from a nucleus at anaphase. (B, IV) Plane from a nucleus at late anaphase. (B, V) Plane from a nucleus at early telophase. (B, VI) Plane from a nucleus at late telophase. [Scale bars: A, I–IV, and IX, 5 μm (20.5 μm); A, V–VIII, 5 μm (19 μm); B, 5 μm.]