We present a detailed protocol for mouse embryo culture and imaging that enables 3D + time imaging of cardiac progenitor cells. This video-toolkit addresses the key skills required for successful live imaging otherwise hard to acquire from text-only publications.
The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail specific developmental stages in a still snapshot, live imaging captures dynamic morphogenetic events, such as cell migration, shape changes, and differentiation, by imaging the embryo as it develops. This complements fixed analysis and expands the understanding of how organs develop during embryogenesis. Despite its advantages, live imaging is rarely used in mouse models because of its technical challenges. Early mouse embryos are sensitive when cultured ex vivo and require efficient handling. To facilitate a broader use of live imaging in mouse developmental research, this paper presents a detailed protocol for two-photon live microscopy that allows long-term acquisition in mouse embryos. In addition to the protocol, tips are provided on embryo handling and culture optimization. This will help understand key events in early mouse organogenesis, enhancing the understanding of cardiovascular progenitor biology.
The heart forms early during embryogenesis to start pumping nutrients to the whole embryo, while it continues developing1. In mouse embryos, one and a half days after the initiation of gastrulation, a rudimentary heart organ assembles at the anterior pole2,3. By Early Streak (ES) stage, cardiac progenitors in the epiblast ingress through the primitive streak to the nascent mesodermal layer4,5,6 and start migrating to the anterior pole, where they differentiate to form the primitive heart tube. Throughout this process, early heart progenitors undergo cell rearrangements, shape transformations, and differentiation, in addition to migration7 (Figure 1).
Early cardiac progenitors have attracted researchers for nearly a century due to their remarkable ability to differentiate and build a functional organ simultaneously. Over the last two decades, clonal analysis and conditional knockout models have shown that early heart development implicates distinct cell sources in a highly dynamic process8,9,10. However, the 3D structure of the primitive heart tube and the dynamic nature of its morphogenesis make it challenging to study (Figure 1), and we are far from understanding its full complexity11.
To study these dynamic cellular processes, live imaging methods now offer an unprecedented detail7,12,13,14. In the mouse model, live approaches have been key to interrogating developmental topics that are difficult to address by static analysis7,13,15. While long-term ex vivo culture and robust microscope setups are advancing fast16,17, few researchers have the expertise to successfully image live embryos. Although paper-based publications provide enough technical details to reproduce live imaging experiments, some skills and tricks are hard to grasp without visual examples or peer-to-peer assistance. To accelerate this learning process and spread the use of live imaging among laboratories, we assembled a video protocol (Figure 2) that gathers the necessary skills to perform live imaging on gastrulating mouse embryos.
Figure 1: Early differentiation of cardiac progenitor cells in the mouse embryo from the onset of gastrulation to the stage preceding primitive heart tube formation. Cardiac progenitor cells ingress the mesoderm soon after the start of gastrulation, migrating to the opposite side of the embryo. Morphological and embryonic day (E) stage are written on top of the diagrams. Dashed arrows depict the migration trajectory of primitive heart tube progenitors during gastrulation. This figure was adapted from11. Abbreviations: ES = Early Streak; MS = Middle Streak; EHF = Early Head Fold. Please click here to view a larger version of this figure.
Figure 2: Workflow diagram for live imaging of early heart progenitors. Please click here to view a larger version of this figure.
All animal procedures were approved by the CNIC Animal Experimentation Ethics Committee, by the Community of Madrid (Reference PROEX 220/15) and conformed to EU Directive 2010/63EU and Recommendation 2007/526/EC regarding the protection of animals used for experimental and other scientific purposes, enforced in Spanish law under Real Decreto 1201/2005.
This protocol includes the use of two males from the fluorescent transgenic mouse line reporting NOTCH activity Tg(CBF:H2BVenus,+)18. See the Table of Materials for details about materials, animals, and equipment used in this protocol.
1. Tools and holder customization
2. Media preparation
3. Embryo dissection
4. Microscope preparation
5. Embryo mounting
6. Image acquisition
Figure 3: Live imaging tools and setup. (A) Dissecting mouse embryos with an agarose-coated dish under the stereomicroscope. (B) Diagram of the steps to dissect mouse embryos for live imaging. (C) Embryo positioning in the holder. (D) Embryo holder design and features. (F) Incubator chamber around the immersion objective. (G) Diagram of final setup for time-lapse acquisition. Scale bar = 500 µm (A). Please click here to view a larger version of this figure.
We used the protocol to visualize NOTCH signaling activation in early cardiac progenitors about to differentiate to endothelial cells during primitive heart tube morphogenesis. For that, we crossed wild-type C57BL/6-N mice with Tg(CBF:H2BVenus,+) mice18 to obtain embryos reporting NOTCH activity through yellow fluorescent protein Venus. At E7.5, Venus fluorescence is present throughout the neural ectoderm, with a few positive nuclei at the splanchnic and extraembryonic mesoderm. After 4 h, endothelial progenitors activate Venus and assemble to form the endocardial tube and underlying aortas, which become enclosed structures after 7 h (Figure 4).
Figure 4: Live whole embryo heart development by multiphoton imaging. Selected time points from a time-lapse video of a CBF:H2BVenus+/- mouse embryo at early bud stage (E7.5). (A) Brightfield optical sections. (B) 480-510 nm detector band showing Venus fluorescent protein expression. Scale bars = 100 µm. Yellow arrowheads point to the developing endocardial lumen and aortas. The time is indicated in h:min at the top right of each image. Please click here to view a larger version of this figure.
Early heart progenitors organize in a primitive heart tube that starts beating while it is still forming. Understanding how this process takes place is key to pinpoint the wide spectrum of congenital heart defects to specific morphogenetic events. For that, live imaging offers an opportunity to study normal and defective embryonic development with increased temporal resolution. This is especially useful to study early cardiac progenitor cells as they transition quickly through multiple differentiation and migration behaviors7.
Because we can study different embryonic stages in a single specimen, the live imaging protocol presented here can be used to corroborate the interpolation of morphogenetic events studied in static analysis. This will help understand the sequence of developmental events in a continuous manner. Moreover, live analysis yields multi-dimensional data, which allows the classification of cell types with unprecedented precision by measuring cellular behaviors28.
Despite its advantages, the method proposed here has some limitations. Although the embryos can develop well up to 48 h inside the microscope, the region of interest tends to move out of frame in long acquisitions. To prevent that from happening, the user must regularly check the drift of the embryo during the acquisition process. Although automatic systems to correct sample focus exist12, these cannot be implemented in most commercially available microscopes. Alternatively, two or more users can take turns to supervise and adjust the embryo frame and stack, so that long acquisitions are not as challenging. However, it must be kept in mind that long acquisitions have a considerable chance of failure, especially for untrained users. Minor embryo damage during dissection and poor positioning in the holder are two of the main causes. To improve these skills, one can start by culturing mounted embryos in the incubator before trying live imaging experiments. That way, one can test whether embryos develop properly before spending microscopy resources.
In summary, live imaging methods are opening new avenues to study embryonic development. The protocol presented here allows analyzing in detail the origin of phenotypes in mutant embryos or testing the function of specific molecular pathways through pharmacological manipulation. With this video toolkit, we aim to facilitate its broad use toward understanding the complexity of early cardiac progenitors.
The authors have nothing to disclose.
The authors acknowledge Dr. Kenzo Ivanovitch for previous work on this method and the group of Dr. Shigenori Nonaka (National Institutes of Natural Sciences, Japan) for providing the initial expertise on embryo mounting. This study was supported by Grant PGC2018-096486-B-I00 from the Spanish Ministerio de Ciencia e Innovación and Grant H2020-MSCA-ITN-2016-722427 from the EU Horizon 2020 program to MT and Grant 1380918 from the FEDER Andalucía 2014-2020 Operating Program to JND. MS was supported by a La Caixa Foundation PhD fellowship (LCF/BQ/DE18/11670014) and The Company of Biologists travelling fellowship (DEVTF181145). The CNIC is supported by the Spanish Ministry of Science and the ProCNIC Foundation.
#55 Forceps | Dumont | 11295-51 | |
35 mm Dish with glass coverslip bottom 14 mm Diameter | Mattek | P35G-1.5-14-C | |
35 mm vise table | Grandado | SKU 8798771617573 | |
50 mL tubes | BD Falcon | 352070 | |
Distilled water | |||
DMEM – Dulbecco's Modified Eagle Medium | Gibco | 11966025 | with L-Glutamine, without Glucose, without Na Pyruvate |
Fetal Bovine Serum | Invitrogen | 10438-026 | |
Fluorescent reporter transgenic mice (Tg(CBF:H2BVenus,+) | JAX | ||
Fluorobrite DMEM | ThermoFisher | A1896701 | DMEM for live-cell imaging |
High-vacuum silicone grease | Dow Corning | Z273554-1EA | |
Holder for wires | Perlen Pressen | pwb1 | |
LSM 780 Upright microscope | Zeiss | ||
MaiTai Deepsee far red pulsed-laser tuned at 980 nm | Spectra-Physics | ||
Non Descanned Detectors equipped with the filter sets cyan-yellow (BP450-500/BP520-560), green-red (BP500-520/BP570-610) and yellow-red (BP520-560/BP645-710) |
Zeiss | ||
Obj: 20x water dipping 1.0 NA, long working distance | Zeiss | ||
P1000 and P200 pipettes | |||
Paraffin Oil | Nidacon | VNI0049 | |
Penicillin-streptomycin | Invitrogen | 15070-063 | (the final concentration should be 50 μg/mL penicillin and 50 μg/mL streptomycin) |
Petri dishes 35 mm x 10 mm | BD Falcon | 351008 | |
Pipette tips | |||
Polymethyl methacrylate | Reused from old laboratory equipment | ||
Rat Serum culture embryo, male rats SPRAGUE DAWLEY RjHan SD | Janvier Labs | 9979 | |
Set of 160 mm fines | RS PRO | 541-6933 | |
Standard 1.0 mm glass capillaries | Anima Lab | 1B100F-3 | |
Sterile 0.22 μm syringe filter | Corning | 431218 | |
Sterile 5 mL syringe | Fisher Scientific | 15809152 | |
Tungsten needles | |||
Ultrasonic homogeniser (sonicator) | Bandelin | BASO_17021 |