Here, we present the protocol for 3-D tissue culture of the zebrafish posterior body axis, enabling live study of vertebrate segmentation. This explant model provides control over axis elongation, alteration of morphogen sources, and subcellular resolution tissue-level live imaging.
Vertebrate embryos pattern their major body axis as repetitive somites, the precursors of vertebrae, muscle, and skin. Somites progressively segment from the presomitic mesoderm (PSM) as the tail end of the embryo elongates posteriorly. Somites form with regular periodicity and scale in size. Zebrafish is a popular model organism as it is genetically tractable and has transparent embryos that allow for live imaging. Nevertheless, during somitogenesis, fish embryos are wrapped around a large, rounding yolk. This geometry limits live imaging of PSM tissue in zebrafish embryos, particularly at higher resolutions that require a close objective working distance. Here, we present a flattened 3-D tissue culture method for live imaging of zebrafish tail explants. Tail explants mimic intact embryos by displaying a proportional slowdown of axis elongation and shortening of rostrocaudal somite lengths. We are further able to stall axis elongation speed through explant culture. This, for the first time, enables us to untangle the chemical input of signaling gradients from the mechanistic input of axial elongation. In future studies, this method can be combined with a microfluidic setup to allow time-controlled pharmaceutical perturbations or screening of vertebrate segmentation without any drug penetration concerns.
Metameric segmentation of organisms is widely used in nature. Repeated structures are essential for functionality of lateral organs such as vertebrae, muscles, nerves, vessels, limbs, or leaves in a body plan1. As a result of such physiological and geometric constraints of the axial symmetry, most phyla of Bilateria-such as annelids, arthropods, and chordates-exhibit segmentation of their embryonic tissues (e.g., ectoderm, mesoderm) antero-posteriorly.
Vertebrate embryos sequentially segment their paraxial mesoderm along the major body axis into somites with species-specific intervals, counts, and size distributions. Despite such robustness among individual embryos within a species, somite segmentation is versatile in between vertebrate species. Segmentation happens in a vast regime of time intervals (from 25 min in zebrafish to 5 h in humans), sizes (from ~20 µm in tail somites of zebrafish to ~200 µm in trunk somites of mice) and counts (from 32 in zebrafish to ~300 in corn snakes)2. More interestingly, fish embryos can develop in a wide range of temperatures (from ~20.5 °C up to 34 °C for zebrafish) while keeping their somites intact with proper size distributions by compensating for both segmentation intervals and axial elongation speeds. Beyond such interesting features, zebrafish stays as a useful model organism to study segmentation in vertebrates due to the external, synchronous and transparent development of a plenitude of sibling embryos as well as their accessible genetic tools. Adversely from a microscopy perspective, teleost embryos develop on a bulky spherical yolk, stretching and rounding the gastrulating tissue around it (Figure 1A). In this article, we present a flattened 3-D tissue explant culture for zebrafish tails. This explant system circumvents the spherical constraints of yolk mass, allowing access to high resolution live imaging of fish embryos for somite patterning.
Figure 1: Slide Chamber Explant System for Zebrafish Embryos. (A) Zebrafish embryos have advantages for live imaging, such as the transparency of gastrulating embryonic tissue (blue), but the tissue forms around a bulky spherical yolk mass (yellow) which prevents near-objective, high-resolution imaging in intact embryos. Tail explants can be dissected starting with a microsurgical knife (brown) cut from the tissue anterior of somites (red) and continuing at the border with the yolk posteriorly. (B) Dissected tail explants can be placed on a coverslip (light blue) dorsoventrally; keeping neural tissue (light gray) on top and notochord (dark gray) at the bottom. Please click here to view a larger version of this figure.
This protocol involves use of live vertebrate embryos younger than 1 day post-fertilization. All the animal experiments were performed under the ethical guidelines of Cincinnati Children's Hospital Medical Center; animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee (Protocol # 2017-0048).
1. Embryo collection
2. Tool preparation
3. Sample preparation
4. Live image acquisition
5. Immunostaining of tail explants
NOTE: Tissues grown after various dissection scenarios (elongating, non-elongating, tail bud dissected, half PSM etc.) as flat-mounted tail explants5 can be recovered from slide chambers for further immunostaining quantifications of proteins of interest. Here, we present the protocol used for di-phosphorylated extracellular signal regulated kinase (ppERK) staining of explants as FGF signaling gradient readout.
This protocol enables flat geometric culturing of live zebrafish tail explants. Tissue culture presents three major advantages over whole embryos: 1) control of axis elongation speed, 2) control over various signaling (morphogen) sources by simple dissection, and 3) near-objective, high magnification and high NA live imaging.
Chemically untreated slide chambers allow the tail explant to elongate its major axis (Figure 2A) by the skin ectoderm wrapping around the tissue beneath. When we cultured the explants on the chemically activated slide chambers (with Type I Collagen), the skin ectoderm stretched and adhered to the slide chamber, which halted the axis elongation of the explant. Despite this, the somites continued to segment (Figure 2B, Supplementary Movies S1 and S2). As described in the protocol, axis elongation can also be halted directly by applying physical pressure during the mounting process or mounting explants in a shallower slide chamber. Quantification of axis elongation speed under such physical restraints can be found in our previously published work5.
Figure 2: Control of Axis Elongation in Explants. (A) Explants cultured on a regular slide chamber (left) elongate axially as they keep segmenting new somites. Transmitted light (left, grayscale) and transgenic nuclear marker (right, red) snapshots are shown for 2 h of culture duration. (B) Chemically activating the slide chamber with Type I Collagen before culturing stalls the axial elongation but does not affect somite segmentation speed. Scale bar is 100 µm. Please click here to view a larger version of this figure.
Secondly, explants can be cultured by dissecting out the sources of morphogens to identify instructive information they provide for developmental processes. Here we present three exemplary images showing the effect of dissections on ppERK signaling levels (Figure 3). In the PSM tissue an FGF signaling gradient is established from posterior to anterior (read out by ppERK levels). Only the tail bud tissue actively transcribes fgf86 and forms a source for this gradient with the help of FGF ligand diffusivity5. Tail explants missing the tail bud portion of the tissue after dissection (Figure 3C) results in a shorter ppERK gradient (Figure 3B,3D). Opposingly, a retinoic acid gradient is established from the anterior to the posterior in both the PSM and dorsal neural tissue. Recently formed somites and the anterior end of the PSM express retinoic acid (RA) synthesizing enzymes and act as a source for the RA gradient7. When we dissected out the anterior PSM tissue in the explants (Figure 3E), we still observed a normal extent of the ppERK gradient (Figure 3F) as visualized by immunostaining. A detailed utilization of this strength of explant method can be found in our recent study5.
Thirdly, flat-mounted zebrafish explants are optimal for high resolution live observation of tissue morphogenesis. Here we present a movie (Video 4) taken with a transgenic explant expressing EGFP as a cell membrane marker (false colored with red) and stained with a far-red cell nuclei marker (false colored with cyan). Without further quantification, many processes such as ingression of neuromesodermal progenitors into the tail bud, higher motility of posterior PSM cells as compared to anterior, and epithelialization of somitic boundary cells can directly be observed in the movie.
Figure 3: Immunostaining of Tail Explants. (A) Full PSM explants with intact signaling gradients along the PSM as a control. (B) Regular ppERK gradient is observed in full PSM explants with immunostaining. (C) Tail bud dissected explants are missing a major part of posterior FGF signaling source. (D) A very posteriorly restricted ppERK signal is observed in tail bud dissected explants. (E) Anterior PSM and somitic tissue can be dissected out to remove the sources for possible anterior PSM signaling factors such as RA signaling. (F) Anterior PSM removal does not change the normal extent of ppERK gradient. Tissues were fixed 2 h after explanting for immunostaining protocol. Scale bar is 100 µm. Please click here to view a larger version of this figure.
Movie 1: Axis Elongation and Somite Segmentation in 3D Explants. Widefield transmitted light (top) and nuclear localized GFP (false colored red) epifluorescence (bottom) time lapse images of a regular slide chamber flat-mounted explant. Tail tissue was explanted from embryo at 13 somites stage. Image acquisition is performed on an inverted microscope with 3 min frame intervals. Scale bar is 100 µm. Please click here to download this Movie.
Movie 2: Stalled Axis Elongation on Chemically Activated Slide Chamber. Widefield transmitted light (top) and nuclear localized GFP (false colored red) epifluorescence (bottom) time lapse images of a flat-mounted explant. A 11 somites stage embryo explant was mounted on a slide chamber coated with rat tail collagen solution for 30 min before mounting. Image acquisition is performed on an inverted microscope with 3 min frame intervals. Scale bar is 100 µm. Please click here to download this Movie.
Movie 3: Lateral Mounting of Late-Stage Embryo Explants. Widefield transmitted light (left) and nuclear localized GFP (false colored red) epifluorescence (right) time lapse images of a regular slide chamber lateral-mounted explant. Tail tissue was explanted from embryo at 15 somites stage and tricaine solution was used as anesthetics. Image acquisition is performed on an inverted microscope with 3 min frame intervals. Scale bar is 100 µm. Please click here to download this Movie.
Movie 4: Single Cell Resolution Imaging of Tail Explants. Time-lapse confocal imaging of an explant expressing EGFP as membrane marker (false colored red) and far red stained for nuclei in live (false colored cyan). Average intensity projection from 5 z-layers (10 µm) are shown in the movie over 1 hour. Image acquisition is performed on a GaAsP detector inverted confocal microscope with a 40× apochromatic λS DIC-water immersion 1.15 NA objective lens, with 4 min frame intervals. Scale bar is 100 µm. Please click here to download this Movie.
This article presents a detailed protocol of a tissue culture explant technique we developed and used recently5 for zebrafish embryos. Our technique builds on the previous explant methods in chick8 and zebrafish9,10,11 model organisms. Tail explants prepared with this protocol can survive as long as >12 h in a simple slide chamber, continuing to elongate its major body axis and segmenting somites, until the end of somitogenesis.
Care should be given to keep explant tissue healthy and successfully elongating for long durations. First, the tissue explant should be dissected without damaging the intactness of the posterior tissues. We observed that the skin cells are providing a pouch for neuromesodermal progenitor cells ingressing into the posterior tailbud. If the skin of explants gets peeled off these highly motile cells, leave the tailbud tissue and migrate posteriorly over the coverslip beyond the tissue limits. Second, mediolaterally imbalanced tension of the skin tissue can result in divergent axis elongation, which can bend the axis of the PSM and notochord. A short-slit cut made to the flanking skin cells on both sides of the explant helps to alleviate that concern. Besides the skin-relevant concerns, extra attention should be paid to maintaining the sterility of the growth media and dissection tools for longer duration cultures.
With proper care, the explant culture recapitulates the healthy growth of whole embryos. We observed muscular twitches in the explants beginning from 20 somites stage like in whole embryos12. Although we focused on the PSM tissue for study of somite segmentation, adjacent tissues such as skin ectoderm, neural keel (later neural rod and neural tube), notochord, intermediate and lateral plate mesoderm also remain intact under the described culturing conditions (Figure 1B). This is particularly advantageous for tissues obscured by the yolk that can be imaged in high resolution at ventrally mounted explants, such as notochord, Kupffer’s vesicle and other tissues developing at segmentation stages12. It should be emphasized that the explant system does not grow as fast as whole embryos, and do not segment somites at the same pace as whole embryos. This limitation for the explant system can also alter temporal dynamics of other events unobserved here.
Here, we presented representative results that highlight three major advantages of the tail explant system over whole embryo experiments. We recently utilized this method to untangle instructive roles of axis elongation from morphogen gradients for somite segmentation5. Late advances in light sheet microscopy have made whole embryo live imaging possible13 for several model organisms. But most of these methods still lack proper subcellular resolution and are barely accessible to the broader research community. The tail explant model described here makes subcellular resolution tissue-level live imaging accessible with simple inverted or confocal microscopes. Aside from methodological advantages, such live imaging can provide insights on segmentation of the posterior vertebrate body axis. Keeping the protocol as direct and accessible as possible, the explant system can also benefit the broader zebrafish developmental biology field.
The authors have nothing to disclose.
We thank the AECOM Zebrafish Core Facility and Cincinnati Children's Veterinary Services for fish maintenance, the Cincinnati Children's Imaging Core for technical assistance, Didar Saparov for assistance with video production and Hannah Seawall for editing the manuscript. Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R35GM140805 to E.M.Ö. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
1 mL Sub-Q Syringe with PrecisionGlide Needle | Becton, Dickinson and Co. | REF 309597 | for dechorionating embryos and manipulations |
200 Proof Ethanol, Anhydrous | Decon Labs | 2701 | for immunostaining |
Antibiotic Antimycotic Solution (100×) | Sigma-Aldrich | A5955 | for tissue dissection media |
Calcium Chloride Anhydrous, Powder | Sigma-Aldrich | 499609 | for tissue dissection media |
Dimethylsulfoxide | Sigma-Aldrich | D5879 | for immunostaining |
Disposable Scalpel, #10 Stainless Steel | Integra-Miltex | MIL4-411 | for preparing tape slide wells |
Ethyl 3-aminobenzoate methanesulfonate salt (Tricaine) | Sigma-Aldrich | 886-86-2 | (optional) for anesthesizing tissues older than 20 somites stage |
Fetal Bovine Serum (FBS) | ThermoFisher | A3160601 | additional for tissue culture media |
Goat anti-Mouse IgG2b, Alexa Fluor 594 | Invitrogen | Cat#A-21145; RRID: AB_2535781 | secondary antibody for immunostaining |
L-15 Medium with L-Glutamine w/o Phenol Red | GIBCO | 21083-027 | for tissue dissection media |
Methanol | Sigma-Aldrich | 179337 | for immunostaining |
Microsurgical Corneal Knife 2.85 mm Angled Tip Double Bevel Blade | Surgical Specialties | 72-2863 | for tissue dissection |
Mouse monoclonal anti-ppERK | Sigma-Aldrich | Cat#M8159; RRID:AB_477245 | for ppERK immunostaining |
NucRed Live 647 ReadyProbes Reagent | Invitrogen | R37106 | (optional) for live staining of cell nuclei |
Paraformaldehyde Powder, 95% | Sigma-Aldrich | 158127 | for fixation of samples for immunostaining |
Rat Tail Collagen Coating Solution | Sigma-Aldrich | 122-20 | (optional) for chemically activating slide chambers |
Stage Top Incubator | Tokai Hit | tokai-hit-stxg | (optional) for temperature control during live imaging |
Transparent Tape 3/4'' | Scotch | S-9782 | for preparing tape slide wells |
Triton X-100 | Sigma-Aldrich | X100 | for immunostaining |
Tween 20 | Sigma-Aldrich | P1379 | for immunostaining |
Zebrafish: Tg(actb2:2xMCP-NLS-EGFP) | Campbell et al., 2015 | ZFIN: ZDB-TGCONSTRCT-150624-4 | transgenic fish with nuclear localized EGFP |
Zebrafish: Tg(Ola.Actb:Hsa.HRAS-EGFP) | Cooper et al., 2005 | ZFIN: ZDB-TGCONSTRCT-070117-75 | transgenic fish with cell membrane localized EGFP |