We present a protocol for isolating single somites from zebrafish embryos, the dynamics of which can be followed in culture for several hours by fluorescence time-lapse microscopy, thus providing a methodology to quantify tissue-scale shape changes at single-cell resolution.
The body axis of vertebrate embryos is periodically subdivided into 3D multicellular units called somites. While genetic oscillations and molecular prepatterns determine the initial length-scale of somites, mechanical processes have been implicated in setting their final size and shape. To better understand the intrinsic material properties of somites, a method is developed to culture single-somite explant from zebrafish embryos. Single somites are isolated by first removing the skin of embryos, followed by yolk removal and sequential excision of neighboring tissues. Using transgenic embryos, the distribution of various sub-cellular structures can be observed by fluorescent time-lapse microscopy. Dynamics of explanted somites can be followed for several hours, thus providing an experimental framework for studying tissue-scale shape changes at single-cell resolution. This approach enables direct mechanical manipulation of somites, allowing for dissection of the material properties of the tissue. Finally, the technique outlined here can be readily extended for explanting other tissues such as the notochord, neural plate, and lateral plate mesoderm.
Much of the vertebrate adult musculoskeletal system emerges from embryonic somites, which form in a periodic and rhythmic manner along the body axis of embryos1,2. Somites are three dimensional (3D) multicellular units typically consisting of an inner core of mesenchymal cells and a peripheral epithelial layer surrounded by a fibronectin-rich extracellular matrix3. The morphology of somites i.e., their size and shape, is in part determined by the segmentation clock and downstream molecular prepatterns. However, over the last decade, it has emerged that mechanical cues and forces also play a role in regulating the segmentation clock4, in addition to facilitating somite formation5,6,7, and ensuring increased precision of somite lengths following initial somite formation8.
Tissue mechanics can be studied directly in vivo with the availability of new tools9, however, to obtain a complete picture underlying the physical processes, the intrinsic material properties of tissues need to be simultaneously studied. The protocol described here provides a simple approach to prepare single somites, whose physical properties from cellular to tissue scales can be studied in isolation from the embryo. While several protocols exist for preparing explants at similar developmental stages10,11,12,13,14, to our knowledge, this is the first protocol that describes isolation of single somites. The protocol is straightforward to implement and requires only basic equipment available in most zebrafish labs working with embryos.
To aid in teasing apart the role of mechanics in morphological somite formation, a method is developed to culture single-somite explant from zebrafish embryos, which can be used to probe intrinsic material properties of somites.
This protocol involves use of live vertebrate embryos younger than 1-day post-fertilization. All experiments were carried out using embryos derived from freely mating adults, and thus are covered under the general animal experiment license of the EPFL granted by the Service de la Consommation et des Affaires Vétérinaires of the canton of Vaud – Switzerland (authorization number VD-H23).
1. Before dissection
2. Single-somite explant preparation
3. Imaging single-somite explants
NOTE: Here, the procedure for imaging single-somite explants using a single-view light-sheet microscope is described. As an alternative, the explanted single somites can also be imaged in a confocal microscope, which is more widely available or even in a widefield microscope if exclusively interested in following overall morphology of explants.
Explants allow for quantification of large-scale shape changes in 3D. To illustrate this, we explanted somite four (N = 3) from early zebrafish embryos obtained from a cross between Utr::mCherry (Tg(actb2:mCherry-Hsa.UTRN); e119Tg) and H2B::GFP (Tg(h2az2a:h2az2a-GFP); kca6Tg) heterozygous lines. Utrophin is an actin-binding protein, and the Utr::mCherry line shows the distribution of filamentous actin structures. This transgene was used here as a marker for cell outlines. H2B::GFP is a fluorescently-tagged histone and consequently marks the distribution of the chromatin, providing an effective description of nuclear location and shape, as well as mitotic figures that highlight cell division.
We assessed the success of the explant protocol during imaging. We observed that in a somite damaged during dissection either the integrity of the somite tissue is compromised, with many cells dissociating and extruding from the explanted somite, and/or many cells dying, which can be noted by the presence of fragmented nuclei in the nuclear channel. Successful explants remained healthy for 4-6 h after which changes in somite integrity were observed with cells dissociating from the explant and dying.
In the utrophin channel, we performed manual segmentation of explants in MATLAB (R2018b) on z-slices spaced every 10 µm. We developed a custom algorithm in MATLAB for segmentation, which is freely available for download (https://github.com/sundar07/SomSeg). In the algorithm, the file, frame and z-slice to be segmented can be set, followed by a user prompt, which allows for manual drawing of an outline around the region of interest. This was repeated for multiple z-slices and plotted, which showed rounding up of explants over time in 3D (Figure 3A). In addition, complementary information on tissue shape using the nuclear channel was also obtained. For this, we used Mastodon (version 1.0.0-beta-19, https://github.com/mastodon-sc/mastodon), a FIJI15 plugin, to obtain nuclei centroid positions through spot detection. We first converted the tif files from the microscope to xml/hdf5 format in FIJI, following which a new project was opened using the Mastodon plugin. In the plugin, we chose the spot detection option, where we defined a region of interest that covered the explant and used the difference of Gaussian detector with a diameter of 5 µm and a quality factor of 25 for detecting spots. We then transferred the nuclei centroid positions to MATLAB and used an in-built function (convhull) to obtain a convex hull (Figure 3B), which characterized the geometry of the explant. This similarly showed rounding of explants over time (Figure 3B). The nuclei data additionally allows for quantifying cell movements by tracking in 3D and a change in cell number over time. On the other hand, the utrophin channel allows for quantification of changes in cell shapes as explants become rounder. Together, these parameters are valuable for characterizing intrinsic material properties of somites, which aids in developing effective physical descriptions of tissue-scale shape changes.
Explants also allow for characterizing contact stresses with neighboring tissues in a quantitative manner. To illustrate this, we manually isolated two somites and placed them in close proximity and observed their dynamics (N = 2). For this experiment, the orientation of the somites with respect to the in vivo body axes was not tracked. Interestingly, over time, the explanted somites adhered to each other along one surface, while the free surfaces i.e., regions away from the contact site rounded up (Figure 4). This suggests that adhesive forces overcome stresses generated by surface tension at contact sites in explants. This can be further characterized by following shape changes as described above and by quantifying contact angles between the two tissues over time in 3D. Thus, the explants provide an attractive system for quantifying competing forces in action that lead to specific tissue shapes, the implications of which can then be explored in vivo.
Figure 1: Preparation of single-somite explants. Zebrafish embryo (A) is first dechorionated (B), followed by removal of skin and yolk (C). Somite-containing region of the embryo is then selected by removing rest of the tissues (D, E). Regions around the somite of interest are then serially removed (F–H) to ultimately isolate a single somite (I), followed by time-lapse imaging using a light-sheet microscope (middle z-section of somite shown here) (J). Abbreviations: S = somite; N = notochord; LPM = lateral plate mesoderm; A = anterior; P = posterior. Dashed lines indicate cut positions. Scale bar = 50 µm in all panels. Please click here to view a larger version of this figure.
Figure 2: Assembly of light-sheet imaging chamber. (A–D) A FEP membrane strip (dimensions in (B)) is wrapped around the membrane holder, fitted on to the imaging chamber and the membrane is glued to the imaging chamber. (E–F) The following day, the membrane holder is removed and an imaging mold (dimensions in (E)) is placed in the center of the imaging chamber in low-melting agarose. The entire unit is kept at 4 °C for 30 min, following which the mold is removed and the chamber with the trough is used for imaging explants. Please click here to view a larger version of this figure.
Figure 3: 3D analysis of tissue shapes. (A) Cell outlines were visualized with fluorescently-tagged utrophin (Utr::mCherry) and nuclei were visualized with fluorescently-tagged histone (H2B::GFP). The outlines of the explant were manually segmented at multiple depths using MATLAB and shown here. (B) Nuclei (red) were detected in the same explant using Mastodon, a FIJI plugin, and subjected to a convex hull (cyan), which informs on the geometry of the explant. Note rounding up of explants is evident in both analyses. Please click here to view a larger version of this figure.
Figure 4: Explanted somites adhere to each other. Two somites isolated from embryos and manually placed in close proximity tend to adhere over time (N = 2). Multiple z-slices with a frame interval of 2 min were acquired using a light-sheet microscope and middle sections of somites from selected time points are shown here. Cell outlines were visualized with fluorescently-tagged Utrophin (Utr::mCherry). Scale bar = 25 µm. Please click here to view a larger version of this figure.
The somitogenesis field has been dominated by studies of the segmentation clock's role in setting segment lengths during embryo development. However, its equally important to consider the role of tissue mechanics in determining final somite morphologies. The protocol described here allows for explanting single somites, whose intrinsic physical properties can be studied in isolation from the embryo. However, the manual preparation limits the numbers of somites that are typically prepared to four to six per imaging session. Since the protocol involves careful fine dissections of several tissues in embryos without the use of any enzymes to facilitate tissue removal, it may take a few weeks of practice to master the dissection process. In our hands the critical steps in the protocol involve careful removal of skin and yolk from the explants, which prevents explants at different stages of the protocol from sticking to the tools used, which in turn enables easier dissections to serially remove tissues surrounding a somite.
The dissection protocol can be stopped at intermediate steps to obtain somites attached to just one of the surrounding tissues or to obtain explants of groups of somites. This provides a powerful method to serially strip down tissues and study the impact of neighboring tissues in facilitating shape changes in somites, or in the neighboring tissues. On the other hand, using this protocol, individual explanted tissues can be allowed to adhere and mechanically self-organize, as demonstrated by placing two explanted somites in close proximity.
Explants prepared by this method do not require any added ingredients in the buffer or constraints to ensure survival for periods of time up to several hours. However, one can envision culturing them in hydrogels and following their dynamics in the presence of external constraints, which could serve as a model for the contact stresses somites encounter in vivo. Furthermore, explants allow for directly probing their material properties through atomic force microscopy, pipette aspiration, or by using micro-robotic tools16. Finally, we expect that this method can be easily adapted towards culturing and studying other developmental tissues at similar stages such as the notochord, neural plate and lateral plate mesoderm.
The authors have nothing to disclose.
We thank members of the Oates lab for comments on the protocol and the fish facility of the École polytechnique fédérale de Lausanne (EPFL). In particular, we acknowledge Laurel Ann Rohde for valuable tips on skin and yolk removal in the dissection protocol; Arianne Bercowsky Rama for building an efficient pipeline for processing light-sheet data sets through Mastodon; Jean-Yves Tinevez for building the open-source Mastodon software; Marko Popović for tips on data analysis; Chloé Jollivet, Guillaume Valentin and Florian Lang for extensive support in the fish facility; Petr Strnad and Andrea Boni for building the light-sheet microscope and for tips on light-sheet imaging. This work was supported by EPFL and S.R.N. was supported by a Long-Term Human Frontier Science Program postdoctoral fellowship (LT000078/2016).
Agarose | Sigma | 9012-36-6 | For coating bottom of petri dishes |
Agarose, low gelling temperature | Sigma | 39346-81-1 | For preparing Viventis imaging chamber |
Camera | Andor | Andor Zyla 4.2 Plus | For image acquisition in the light-sheet microscope |
Detection objective | Nikon | Nikon CFI75 Apo LWD 25x/1.1 NA | For imaging explants |
FEP membrane strip | Lohmann Technologies UK Ltd | Dupont FEP Fluorocarbon film, 200A | For preparing Viventis imaging chamber |
Fine forceps | Dumont | Dumont 5SF 11252-00 | For removal of skin of embryos |
Forceps | Dumont | Dumont 55 | For dechorionating embryos |
Leibovitz's L-15 medium | Gibco | 21083-027 | Explant culture medium |
Light-sheet microscope | Viventis | LS1 live | For imaging explants |
Micro knife | Fine Science Tools | 10318-14 | For making incisions in embryos |
Silicone rubber formulation | Wacker Chemie AG | Silpuran 4200 | For preparing Viventis imaging chamber |
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