Here, we describe computational tools and methods that allow visualization and analysis of three and four-dimensional image data of mouse embryos in the context of axial elongation and segmentation, obtained by in toto optical projection tomography, and by live imaging and whole-mount immunofluorescence staining using multiphoton microscopy.
Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a wide range of techniques encompassing ex vivo and in vitro approaches. However, most studies still rely on the analysis of two-dimensional (2D) imaging data, which limits proper evaluation of a developmental process like axial extension and somitogenesis involving highly dynamic interactions in a complex 3D space. Here we describe techniques that allow mouse live imaging acquisition, dataset processing, visualization and analysis in 3D and 4D to study the cells (e.g., neuromesodermal progenitors) involved in these developmental processes. We also provide a step-by-step protocol for optical projection tomography and whole-mount immunofluorescence microscopy in mouse embryos (from sample preparation to image acquisition) and show a pipeline that we developed to process and visualize 3D image data. We extend the use of some of these techniques and highlight specific features of different available software (e.g., Fiji/ImageJ, Drishti, Amira and Imaris) that can be used to improve our current understanding of axial extension and somite formation (e.g., 3D reconstructions). Altogether, the techniques here described emphasize the importance of 3D data visualization and analysis in developmental biology, and might help other researchers to better address 3D and 4D image data in the context of vertebrate axial extension and segmentation. Finally, the work also employs novel tools to facilitate teaching vertebrate embryonic development.
Vertebrate body axis formation is a highly complex and dynamic process occurring during embryonic development. At the end of gastrulation [in the mouse, around embryonic day (E) 8.0], a group of epiblast progenitor cells known as neuromesodermal progenitors (NMPs) become a key driver of axial extension in a head to tail sequence, generating the neural tube and paraxial mesodermal tissues during neck, trunk and tail formation1,2,3,4. Interestingly, the position that these NMPs occupy in the caudal epiblast seems to play a key role in the decision of differentiating into mesoderm or neuroectoderm5. Although we currently lack a precise molecular fingerprint for NMPs, these cells are generally thought to co-express T (Brachyury) and Sox25,6. The exact mechanisms regulating NMP fate decisions (i.e., whether they take neural or mesodermal routes) are only starting to be precisely defined. Tbx6 expression in the primitive streak region is an early marker of NMP fate decision, as this gene is involved in the induction and specification of mesoderm6,7. Interestingly, early mesoderm cells seem to express high levels of Epha18, and Wnt/β-catenin signalling, as well as Msgn1 were also shown to play important roles in paraxial mesoderm differentiation and somite formation9,10. A complete spatial-temporal analysis of NMPs at a single-cell level will certainly be instrumental to fully understand the molecular mechanisms controlling mesoderm specification.
The formation of somites (vertebrae precursors) is a key feature of vertebrates. During axial elongation, the paraxial mesoderm becomes segmented in a series of bilateral repeating units known as somites. The number of somites and the time required for the formation of new segments varies among species11,12. Somitogenesis involve periodic signaling oscillations (known as the "segmentation clock") that can be observed by the cyclic expression of several genes of the Notch, Wnt and Fgf signalling pathways in the presomitic mesoderm (e.g., Lfng)11,12. The current model of somitogenesis also postulates the existence of a "maturation wavefront", a series of complex signalling gradients involving Fgf, Wnt and retinoic acid signaling that define the position of the posterior border of each new somite. A coordinated interaction between the "segmentation clock" and the "maturation wavefront" is therefore fundamental for the generation of these vertebrae precursor modules as perturbations in these key morphogenetic processes can result in embryonic lethality or in the formation of congenital malformations (e.g., scoliosis)13,14,15.
Despite substantial recent advances in imaging techniques, bioimage analysis methods and software, most studies of axial elongation and somitogenesis still rely on single/isolated two-dimensional image data (e.g., sections), which does not allow a full multidimensional tissue visualization and complicates clear differentiation between pathological malformations (i.e. due to mutations) vs normal morphological variation occurring during embryonic development16. Imaging in 3D has already uncovered novel morphogenetic movements, previously not identified by standard 2D methods17,18,19,20, highlighting the power of in toto imaging to understand the mechanisms of vertebrate somitogenesis and axial extension.
3D and 4D microscopy of mouse embryos, particularly live imaging, are technically challenging and require critical steps during sample preparation, image acquisition and data pre-processing in order to allow accurate and meaningful spatio-temporal analysis. Here, we describe a detailed protocol for live imaging and whole-mount immunofluorescence staining of mouse embryos, that can be used to study both NMPs and mesodermal cells during axial extension and segmentation. In addition, we also describe a protocol for optical projection tomography (OPT) of older embryos and fetuses, that allows 3D in toto visualization and quantification of pathological abnormalities that can result from problems during somitogenesis (e.g., bone fusion and scoliosis)13,21,22. Finally, we illustrate the power of 3D imaging reconstructions in the study and teaching of vertebrate segmentation and axial elongation.
Experiments involving animals followed the Portuguese (Portaria 1005/92) and European (Directive 2010/63/EU) legislations concerning housing, husbandry, and welfare. The project was reviewed and approved by the Ethics Committee of 'Instituto Gulbenkian de Ciência' and by the Portuguese National Entity, 'Direcção Geral de Alimentação e Veterinária' (license reference: 014308).
1. Sample preparation for 3D and 4D imaging
NOTE: Here we provide a detailed description on how to dissect and prepare mouse E8.25 to E10.5 embryos for live imaging (1.1), E7.5 to E11.5 embryos for whole mount immunofluorescence microscopy (1.2) and fetuses for optical projection tomography (1.3).
Developmental stage | Recommended fixation time (PFA 4%) |
E7.5 | 1h30 |
E8.5 | 2h |
E9.5 | 3h |
E10.5 | 4h |
E11.5 | 4h |
2. Microscope/Image acquisition
Optical microscopy techniques | Imaging principle | Experimental goal and considerations |
Widefield imaging | Uses fluorescence, reflected or transmitted light. | Ideal for a quick and general overview of the embryo (e.g. for screenings and to assess developmental stages and obvious phenotypes). The reduced depth-of-field, compared to the observable thickness at high magnifications does not allow accurate interpretation or analysis of 3D morphology. |
Confocal (laser scanning; CLSM) | Uses laser scanning illumination and detection of fluorescence through a pinhole. | Allows imaging of optical slices of fluorescently-labelled samples, ideally with a strong signal. Imaging through a pinhole removes the signal from out of the depth-of-field, thereby allowing accurate discrimination of information in 3D. The acquisition is orders of magnitude slower than widefield, but with unparalleled contrast and 3D discrimination of morphology. High temporal resolution is not achievable because images are acquired 1-pixel at a time. Good for 3D imaging of fixed mouse embryos up to E9.5. Imaging through the whole presomitic mesoderm or somites requires tissue-clearing, because of light-scattering in deeper tissues. Phototoxicity and bleaching is a consideration. Photobleaching can, within limits, be compensated a posteriori. However, phototoxic effects in live samples cannot, and often are not easy to determine. This is more obvious if samples show low expression, and high-laser powers are needed. |
Two-photon excitation fluorescence (TPEFM) | It uses pulsed near-infrared (NIR) laser excitation instead of visible laser illumination. | TPEFM allows optical slicing through thicker samples than CLSM. Resolution is slightly lower, but the contrast in deeper tissues is considerably better, making it ideal for live imaging of mouse embryos. Although TPEFM is often considered less phototoxic than conventional CLSM, it requires high laser powers which may also have deleterious effects on cells and tissues. Ideal for 3D imaging of embryos up to E11.5, although imaging through the whole presomitic mesoderm still requires tissue-clearing. |
Confocal (spinning disk) | A form of confocal which, instead of a single laser, uses multiple point-like sources. | The use of multiple point-like structures allows faster creation of optical slices (several frames per second or stacks per minute are achievable) than CLSM. The acquisition is practically as fast as widefield, with reasonable 3D discrimination. However, it only allows imaging of the most superficial tissues of the mouse embryo. Allows more sensitive detection than CLSM, making it an alternative for embryos with low expression of fluorescence proteins. |
Light-sheet / single plane (LSFM/SPIM) | Instead of widefield or point-like illumination, the sample is illuminated one orthogonal plane at a time. In most configurations, it allows imaging from multiple angles. | Also requires fluorescently-labelled samples. Acquisition of optical slices acquisition is extremely fast (multiple frames per second) and has reduced effects of phototoxicity or bleaching. However, if multiview is required, subsequent dataset pre-processing steps may require hours/days of computation. LSFM/SPIM allows better detection of lower expression levels than CLSM. Ideal for 3D imaging of in toto mouse embryos during gastrulation. Samples often need to be mounted and maintained in suspension (unconventional preparation). |
Optical projection tomography (OPT) | Optical slices are not detected but calculated from a series of widefield images of the whole embryo from different angles (the “projections”). | Ideal for 3D imaging of later stage mouse embryos/fetuses (>5mm), but only fixed and cleared. Has the advantage of producing 3D stacks of optical slices of both fluorescent and non-fluorescent samples. Datasets are isometric (slices with equal resolution in all three dimensions) making it ideal for anatomical analysis. Acquiring a projection dataset may require only a few minutes, followed by 15-30 min of reconstruction. |
Optical Coherence Tomography (OCT) | Uses NIR illumination through the sample to obtain optical slices based on interference with light reflection. | OCT allows easy imaging through live tissue (a few millimeters deep into the sample without fluorescent contrast) with a few dozen micrometers resolution. The acquisition is very fast (a few slices per second). Although it is a possible alternative for OPT, this technique is not commonly available. |
Super-resolution (SR), atomic force (AFM) or near-field imaging (NSOM) | SR is normally based on single-molecule localization and AFM/NSOM on scanning surfaces at sub-diffraction resolutions (few nanometers). | Allows imaging at sub-diffraction level (<200nm resolution), often with the intent to detect single molecules or molecules at the cell surface. Not ideal for morphological analysis of large samples such as mouse embryos. The acquisition is typically a slow process (seconds to minutes per image). |
Table 2 – Generic information to guide the selection of the imaging technique/microscope more suitable for the researcher's specific experimental goal.
3. Image dataset pre-processing
NOTE: Here we highlight some of the key steps of image dataset pre-processing, namely noise reduction (3.1) and deconvolution (3.2), and provide algorithms that allow proper preparation and pre-processing of 3D datasets time-series (3.3) and whole-mount immunofluorescence stainings (3.4). Finally, we indicate references that describe in detail a protocol for OPT dataset pre-processing and reconstruction.
4. 3D rendering, visualization and analysis
NOTE: Here we provide a list of possible applications of different software tools, that allow or enhance the visualization and analysis of 3D imaging datasets.
The representative results shown in this paper for both the live and the immunofluorescence imaging, were obtained using a two-photon system, with a 20 × 1.0 NA water objective, the excitation laser tuned to 960 nm, and GaAsP photodetectors (as described in Dias et al. (2020)43. Optical projection tomography was done using a custom built OPenT scanner (as described in Gualda et al. (2013)28.
Live imaging (4D analysis)
A representative analysis of LuVeLu reporter activity in mouse embryos during axial extension, obtained according the described protocols for "Sample preparation for live imaging" (Step 1.1; "Sample preparation for 3D imaging" section), "Live imaging dataset pre-processing" (Step 3; "Image dataset pre-processing" section) and "3D visualization and analysis using Imaris" (Step 4.4; "3D rendering, visualization and analysis") can be observed in Figure 1 and Video 1.
3D visualization and analysis
A representative result for the use of immunofluorescence assays to detect potential NMPs and key regulators of mesoderm differentiation, obtained following the protocols described for "Sample preparation for immunofluorescence microscopy" ("Sample preparation for 3D and 4D imaging" section, step 1.2), "Deconvolution" and "Immunofluorescence imaging dataset pre-processing" ("Image dataset preprocessing" section, step 3.2 and 3.4 respectively), and "3D visualization and analysis using Imaris" ("3D rendering, visualization and analysis", step 4.4) can be observed in Figure 2.
3D renderization of immunofluorescence stainings
Video 2 shows a representative result for an immunofluorescence assay to detect potential NMPs obtained according to the described protocols for "Sample preparation for immunofluorescence microscopy" (Step 1.2; "Sample preparation for 3D and 4D imaging" section), the "Deconvolution" and "Immunofluorescence imaging dataset pre-processing" (Step 3.2 and 3.4, respectively; "Image dataset pre-processing" section), and the "3D rendering and visualization using Drishti" (Step 4.1; "3D rendering, visualization and analysis" section).
3D reconstructions
Video 3 was generated following the described methods for "Sample preparation for immunofluorescence microscopy" (Step 1.2; "Sample preparation for 3D and 4D imaging" section), "Immunofluorescence imaging dataset pre-processing" (Step 3.4; "Image dataset pre-processing" section), and "3D reconstructions and manual segmentation of tissues using Amira" (Step 4.2; "3D rendering, visualization and analysis"). It shows a 3D reconstruction, based on immunofluorescence staining, of the caudal tissues of a E9.5 wild type (WT) mouse embryo.
Interactive 3D illustrations
In Figure 3 we present an interactive 3D visualization of a reconstruction of the caudal tissues of mouse embryos in a portable document format (3D PDF), prepared following the described protocols for "Sample preparation for immunofluorescence microscopy" (Step 1.2; "Sample preparation for 3D and 4D imaging" section), "Immunofluorescence imaging dataset pre-processing" (Step 3.4; "Image dataset preprocessing" section), "3D reconstructions and manual segmentation of tissues using Amira", and "Interactive 3D visualization within in a portable document format (PDF) using SimLab" ("3D rendering, visualization and analysis", Step 4.2 and 4.3 respectively). This type of format can be easily used to facilitate communicating scientific contents to a more general audience, particularly in a teaching environment. Interactive visualization requires the use of Adobe Acrobat Reader (allow the 3D plugin).
OPT dataset visualization
Video 4 shows a representative example of an OPT reconstructed dataset, after imaging a E18.5 mouse fetus. Sample was prepared as described in protocols for "Sample preparation for optical projection tomography" ("Sample preparation for 3D and 4D imaging" section; Step 1.3) the "OPT dataset pre-processing and reconstruction" (Step 3.5) and different modules of the "3D rendering, visualization and analysis" (Step 4). A detailed anatomical analysis (e.g., measurements) can also be performed in this dataset using Fiji/ImageJ, Amira or the Imaris software. The movie contains four video segments: the first shows a sequence of black and white sagittal slices produced with Fiji/ImageJ; the second, orthogonal views produced with Imaris showing interactive 3D slicing and rendering of orthogonal tissue portions; the third shows the fetus "assembling" from colored sagittal slices, as prepared in Amira; the last video segment display an animation of the fetus in 3D from different views, prepared with Drishti.
Video 1 –Two-photon live imaging of LuVeLu reporter expression in Snai1-cKO and control E8.5 embryos (adapted from ref. 43). The first segments show the reporter activity at time = 0 (z-stack of 5 µm step-size) and the second parts contains the full live imaging (10 µm z-stacks every 8.5 min). Please click here to download this Video.
Video 2 – 3D renderization of a whole-mount immunofluorescence staining for T (Brachyury) and Sox2 of a E10.5 wild type tailbud. T expression is shown in magenta, Sox2 in yellow and DAPI in grey. Please click here to download this Video.
Video 3 – 3D reconstruction of the caudal part of a late E9.5 wild type mouse embryo, with a focus on the contouring of different caudal tissues including mesoderm. Please click here to download this Video.
Video 4 – E18.5 wild type embryo as imaged with optical projection tomography, highlighting the in toto imaging and several techniques for visualizing and rendering 3D datasets. The first segment shows a sequence of sagittal slices prepared in Fiji/ImageJ, the second "live" orthogonal views as rendered in Imaris, the third a sequential rendering from colored sagittal slices prepared in Amira, and the fourth an animated rendering prepared in Drishti. Please click here to download this Video.
Figure 1: 4D analysis of LuVeLu reporter expression in E8.5 Snai1-cKO and control embryos (adapted from ref. 43). (A) Snapshot at time-point = 0 of the LuVeLu reporter, in Snai1-cKO(Meox2-Cre+/0::Snai1flox/-) (Aa) and control (Ab) embryos. In addition to the normal LuVeLu signal in the presomitic mesoderm, Snai1-cKO embryos also display LuVeLu expression in the ectopic bulge that arises from the primitive streak (white arrows). (B) Snai1-cKO::LuVeLu+/0 temporal intensity mean quantitative analysis in the region highlighted by the red spot (Ba) indicates the existence of two-peaks (at t = 1.3 h and t = 3.6 h of the time-lapse) and a substantial decrease between them, therefore suggesting cycling activity in the bulge of the conditional mutant embryo. No signs of LuVeLu cycling activity were observed near the primitive streak (green spot; Bb) in LuVeLu+/0 control embryos. Scale bar: 50 µm. Please click here to view a larger version of this figure.
Figure 2: Whole-mount immunohistochemistry in E9.5 Snai1-cKO and WT embryos (adapted from ref. 43). (A) Immunostainings for T (green) and Sox2 (red). (B) Immunostainings for Tbx6 (green) and Lam1 (red). DAPI is shown in blue. 3D rendering (blend mode) of the caudal part of the different embryos (Aa, Ab, Ba, and Bb). Transversal (Aa1, Ab1, Ba1, Ba2, Bb1 and Bb2) and sagittal (Aa2, Ab2, Ba3 and Bb3) optical sections together with magnifications (Mag.) are also shown for the different embryos. Magnifications are shown without DAPI. White arrows highlight differences in the location of some NMPs (T and Sox2 double-positive cells) in the mutant embryos. Yellow arrows and arrowheads point to ectopic/abnormal Tbx6 and Lam1 expression respectively, in the Snai1-cKO embryos. Scale bars correspond to 50 µm. Please click here to view a larger version of this figure.
Figure 3 – 3D PDF – Interactive 3D illustration in 3D PDF format showing the 3D reconstructions of segmented caudal tissues of wildtype mouse embryos during axial extension (E8.5, E9.5 and E10.5). Segmentation was done manually in Amira, and the 3D PDF was assembled and exported in Simlab Composer. Interactive visualization requires Adobe Acrobat Reader (allow the 3D plugin). Please click here to download this figure.
Supplemental Figure 1 – Representative result of a bleached embryo (after Step 3.2; "Sample preparation for 3D and 4D imaging" section). Please click here to download this File.
Supplemental Figure 2 – Representative result of Z-depth signal attenuation (Step 4.1; "Image dataset pre-processing" section). A – before Z-depth signal attenuation; B – good Z-depth signal attenuation; C – incorrect Z-depth signal attenuation. Please click here to download this File.
Supplemental Figure 3 – Representative result of a whole-mount immunofluorescence staining showing and E7.5 mouse embryo that has been repositioned (A) following “Repositioning of embryo to an anatomically standard position using Fiji/ImageJ” (Step 3.4.3; "Image dataset pre-processing" section) versus the same embryo without being repositioned (B). Visualization was obtained using Imaris (see Step 4.1.2; "3D rendering, visualization and analysis" section). T in yellow, Sox2 in magenta and DAPI in blue. Please click here to download this File.
Axial elongation and segmentation are two of the most complex and dynamic processes occurring during vertebrate embryonic development. The use of 3D and 4D imaging with single-cell tracking has been applied, for some time, to study these processes in both zebrafish and chicken embryos, for which accessibility and culture conditions facilitate complex imaging19,44,45,46,47,48,49. In contrast, the mid and late organogenesis stages of the mouse embryo remain poorly studied in such detail, although some advances have been made, for example in intravital imaging50. In this manuscript we provide specific protocols for several methodologies that can be used for the acquisition of multidimensional images and their analysis to facilitate the study of NMPs and their mesoderm derivatives during neck, trunk and tail formation. Although these protocols were designed for mouse embryos, they can be easily tuned to work for explant systems and in vitro models such as 3D embryonic stem cell aggregates like gastruloids51,52. Indeed, if applied to human gastruloids53 these methods would facilitate a better in toto imaging and data analysis of human axial elongation and segmentation, especially given the compatibility of these in vitro model systems with long-term 4D live imaging. In addition to these protocols, we also provide general information about different available microscopy techniques and how they can be used to fit specific experimental goals. We hope this information helps researchers to improve their experimental designs and to take full advantage of the microscopy equipment and image analysis tools available in their laboratories and institutions.
Sample preparation is one of the most important steps of the protocols described in this manuscript, as proper preservation and processing of the embryo during the whole procedure, from dissection to mounting in the microscope, is essential to obtain high quality data. Dehydration (and rehydration), bleaching and clearing procedures are the most critical steps during sample preparation, strongly influencing the final outcome of the imaging. Therefore, we have detailed these steps in our protocol and provided tips that will help researchers to achieve a spotless preparation of their samples. Particularly, we have detailed the use of three different solutions for clearing post-implantation mouse embryos (up to E11.5). Although methyl salicylate and BABB have been used for several years and are efficient clearing reagents, complete laser penetration in some tissues, particularly mesoderm, is sometimes difficult to achieve. Conversely, RapiClear was found to be very effective in clearing mouse embryos at these developmental stages. In our hands, it proved to enable complete laser penetrance in the various embryonic tissues and, since it is not toxic and does not require embryo dehydration, it greatly simplifies the key step of mounting the embryos in the microscopy slide. Also, to overcome the necessity of manipulating the embryos in the slide (cleared embryos become very fragile and difficult to manipulate once they are transparent) or the need to resort to commercial software (e.g., Amira or Imaris), we have provided a simple but efficient pipeline, using Fiji/ImageJ (free open-source software), that allows embryo repositioning to an anatomically standard/specific position during image pre-processing without losing data quality. Therefore, we expect the methods and details that we provide in this manuscript to facilitate and contribute to improve the key step of sample preparation during immunofluorescence assays.
During image acquisition, it is important to consider the configuration of the system, which affects how samples need to be maintained for live imaging or mounted for optimal observation in 3D. For example, mouse embryos require a heating source, high levels of O2 and liquid nutrient-rich media (not solid), making it more difficult to be kept live in a microscope with an upright, or a conventional light-sheet configuration. For correct 3D imaging of embryos, immobilization is also important. Available optics is also another important factor to consider; for proper 3D imaging, high numerical aperture objectives should be preferred however those are not always available, especially when large working distances are required (more than a few hundred micrometers). Physiological (i.e., "dipping") lenses are very common for live imaging however these are not ideal for imaging through a glass-bottom dish or for cleared samples mounted on BABB or methyl salicylate. Although objectives optimized for cleared tissues are becoming more common, they are still relatively rare to find in most laboratories. It is also possible to image cleared tissues with conventional objectives however users must be aware of the limitations and the necessary care before interpreting and analyzing the datasets, as we explain in the sections above. Furthermore, accurate detection and interpretation of imaging results always require careful choice not only of the instrument but also of the operating conditions and setting of parameters. Users are encouraged to read the following reviews 54,55,56,57 to fully understand the principles of proper digital imaging and interpretation of bioimages.
Immunofluorescence applied to (2D) histological sections has been the hallmark of studies seeking to understand tissue and cell organization inside embryos. The emergence of whole-mount immunolabeling methods and non-destructive 3D imaging techniques (as those described here) has provided developmental biologists with the means to investigate tissue and cell spatial organization in intact embryos, where it is easier and more accurate to understand gene and protein expression and their relationship to morphogenetic processes. With in toto imaging, it is now possible to perform virtual histology (virtual sectioning in arbitrary slices; Supplemental Figure 3), and the 3D visualization and analysis tools can be used to easily explore different hypotheses about cell and tissue architecture and communication (see Figure 2, Video 2 and Video 4). Considering that many journals (e.g., eLife and Development) are now giving the opportunity to include videos in the online version of the paper, we urge researchers to take advantage of this opportunity and not only perform 3D and 4D imaging experiments and analysis but also to make 3D videos highlighting their results. This important change in the way data is presented and published, will enable a better comprehension of the results by the researchers and their peers. Ultimately, these methods have the potential to improve our understanding of vertebrate segmentation, particularly regarding the role of NMPs (Attardi et al. (2018)45 and Dias et al. (2020)43 are two good examples).
In this work we have highlighted how the use of some software tools (e.g., Amira and Imaris, but also free open-source tools like Fiji/ImageJ and Drishti) allows and enhances 3D data visualization and analysis in the context of vertebrate axial extension and somitogenesis. In most cases, like for several bioinformatic tools, the software described here can potentially be replaced by another (i.e., there are several software solutions and redundancies). However, we find that the "3D View" function in Imaris allows a 3D visualization with much better quality than the one obtained using the "3D Viewer" Fiji/ImageJ plugin, allowing for example to choose between MIP or blend modes. In addition, we find the "Orthogonal Views" function in Imaris and its pipeline for 3D analysis and quantification (e.g., "Spot" module) user friendlier. In this matter, although practically all software tools allow some form of 3D rendering, we recommend Drishti because of its unique light model and shading which enables the generation of highly realistic renderizations (see last segment of Video 4). The rule of thumb for all bioinformatic processing of image datasets (pre-processing and 3D renderings) is that the end result must remain a faithful representation of what is observed in the embryo. Therefore, we provided simple methods to mitigate the problems created by Z-axis scaling distortion (due to refractive index mismatch) and depth signal attenuation (caused by light scattering in deep samples) which severely affect the quality and interpretation of the image datasets. Using these algorithms, we provided instructions to manually segment and 3D reconstruct mesodermal tissues from whole-mount immunofluorescence stainings, by manual contouring (often not possible with tools of automated segmentation) using the Amira software. We find this software especially useful for manual countouring of tissues where there is no clear physical separation or contrast differences between them (e.g., expression of a specific transcription factor). A non-commercial recommended alternative for Amira, although less powerful, is the LabKIT Fiji/ImageJ plugin (https://imagej.net/Labkit).
We have also included among the representative results an example of a 3D visualization of in toto E18.5 mouse fetus imaged with optical projection tomography, which extends the capabilities of optical imaging and 3D image analysis to the entire mouse embryogenesis22. These methodologies can be used to understand and analyze (e.g., through morphological measurements) skeletal abnormalities, like fused vertebrae, scoliosis or spondylocostal dysostosis, that can later occur due to problems during somitogenesis (e.g., Lfng mutation)13, 58,59,60,61,62. Importantly, this imaging approach allows observation of skeletal malformations in a global context, including other tissues (e.g., muscles) that could also contribute to the phenotypes affecting the vertebral column63.
Finally, and in agreement with recent work highlighting volumetric models made with imaging datasets from the eMouse Atlas Project64, we have described a detailed step-by-step method that can be used both to illustrate the power of 3D models (e.g., 3D PDF) to a more general audience and to help in the study and teaching of vertebrate axial elongation and segmentation. We hope the information presented in this manuscript contributes to changing the way researchers design, analyze and present their experiments, and to improve our knowledge of vertebrate body axis formation.
The authors have nothing to disclose.
We would like to thank Olivier Pourquié and Alexander Aulehla for the LuVeLu reporter strain, the SunJin laboratory for the RapiClear test sample, Hugo Pereira for the help using BigStitcher, Nuno Granjeiro for helping to set up the live imaging apparatus, the IGC animal facility and past and present members of the Mallo lab for useful comments and support during the course of this work.
We thank the technical support of IGC's Advanced Imaging Facility, which is supported by Portuguese funding ref# PPBI-POCI-01-0145-FEDER-022122 and ref# PTDC/BII-BTI/32375/2017, co-financed by Lisboa Regional Operational Programme (Lisboa 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) and Fundação para a Ciência e a Tecnologia (FCT, Portugal). Work described in this manuscript was supported by grants LISBOA-01-0145-FEDER-030254 (FCT, Portugal) and SCML-MC-60-2014 (Santa Casa da Misericórdia, Portugal) to M.M., the research infrastructure Congento, project LISBOA-01-0145-FEDER-022170, and the PhD fellowship PD/BD/128426/2017 to A.D.
Agarose low gelling temperature | Sigma | A9414 | Used to mounting embryos (e.g. for OPT) |
Amira software | Thermofisher | – | Commerial software tool |
Anti-Brachyury (Goat polyclonal) | R and D Systems | AF2085 RRID:AB_2200235 | For immunofluorescence |
Anti-Sox2 (Rabbit monoclonal) | Abcam | ab92494 RRID:AB_10585428 | For immunofluorescence |
Anti-Tbx6 (Goat polyclonal) | R and D Systems | AF4744 RRID:AB_2200834 | For immunofluorescence |
Anti-Laminin111 (Rabbit polyclonal) | Sigma | L9393 RRID:AB_477163 | For immunofluorescence |
Anti-goat 488 (Donkey polyclonal) | Molecular Probes | A11055 RRID:AB_2534102 | For immunofluorescence |
Anti-rabbit 568 (Donkey polyclonal) | ThermoFisher Scientific | A10042 RRID:AB_2534017 | For immunofluorescence |
Benzyl Alcohol (99+%) | (any) | – | Used to clear embryos (component of BABB) |
Benzyl Benzoate (99+%) | (any) | – | Used to clear embryos (component of BABB) |
Bovine serum albumin | Biowest | P6154 | For immunofluorescence |
Coverglass 20×20 mm #0 | (any) | – | 100um thick |
Coverglass 20×20 mm #1 | (any) | – | 170um thick |
Coverglass 20×60 mm #1.5 | (any) | – | To use as “slides” |
DAPI (4’,6-Diamidino-2- Phenylindole Dihydrochloride) | Life Technologies | D3571 | For immunofluorescence |
Drishti software | (open source) | – | Free software tool |
EDTA | Sigma | ED2SS | For demineralization |
Fiji/ImageJ software | (open source) | – | Free software tool |
Glycine | NZYtech | MB01401 | For immunofluorescence |
Huygens software | Scientific Volume Imaging | – | Commerial software tool |
HyClone defined fetal bovine serum | GE Healthcare | #HYCLSH30070.03 | For live imaging |
Hydrogen peroxide solution 30 % | Milipore | 1085971000 | For clearing |
Imaris software | Bitplane / Oxford instruments | – | Commerial software tool |
iSpacers | SunJin Lab | (varies) | Use as spacers for preparations |
L-glutamine | Gibco | #25030–024 | For live imaging medium |
Low glucose DMEM | Gibco | 11054020 | For live imaging medium |
M2 medium | Sigma | M7167 | To dissect embryos |
Methanol | VWR | VWRC20847.307 | For dehydration and rehydration steps |
Methyl salicylate | Sigma | M6752 | Used to clear embryos |
Paraformaldehyde | Sigma | P6148 | Used in solution to fix embryos |
Penicillin-streptomycin | Sigma | #P0781 | For live imaging medium |
PBS (Phosphate-buffered saline solution) | Biowest | L0615-500 | – |
RapiClear | SunJin Laboratory | RapiClear 1.52 | Used to clear embryos |
Secure-Sea hybridization chambers | Sigma | C5474 | Use as spacers for preparations |
simLab software | SimLab soft | – | Commerial software tool |
Slide, depression concave glass – 75×25 mm | (any) | – | To mount thick embryos. |
Triton X-100 | Sigma | T8787 | For immunofluorescence |