Live Imaging of Early Cardiac Progenitors in the Mouse Embryo

Summary

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.

Abstract

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.

Introduction

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 pole^2,3. By Early Streak (ES) stage, cardiac progenitors in the epiblast ingress through the primitive streak to the nascent mesodermal layer^4,5,6 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 process^8,9,10. 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 detail^7,12,13,14. In the mouse model, live approaches have been key to interrogating developmental topics that are difficult to address by static analysis^7,13,15. While long-term ex vivo culture and robust microscope setups are advancing fast^16,17, 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.

Protocol

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

1. Cut 1 cm long tungsten wire pieces and sharpen them to 0.02-0.05 mm diameter using any of the simple methods available, for example, submerging the tip of the wire in a saturated sodium nitrite solution¹⁹.
2. Prepare elbowed glass capillaries.
   1. Place the midpoint of a standard 1.0 mm glass capillary over a Bunsen lighter for 3-6 s, pulling slowly from both ends until the capillary turns thin and flexible. At that point, break the capillary in half and heat it briefly to produce a 90° bend 2 cm from the tip.
3. Saw a piece of polymethyl methacrylate measuring approximately 7 mm long, 4 mm wide, and 2 mm thick (Figure 3D).
   NOTE: Polymethyl methacrylate sheets can be either obtained from recycled laboratory equipment or ordered new.
4. Hold the methacrylate piece using a bench vise. Next, drill custom-sized holes transversally through the whole piece (Figure 3D).
   NOTE: Typically, E6.5 to E7.5 mouse embryos require using 0.2-0.5 mm diameter drills.
   1. Rotate the drill slowly while applying low but constant pressure. If a drill breaks inside the holder, discard the piece, as the metal cannot be taken out.
5. Use a fine file to smooth the holder's edges and adjust its size. Additionally, create a mild slope on the edges of the holder to facilitate embryo positioning (Figure 3D).
6. Place the finished holder in a dish with distilled water to rinse it.
7. Check the holder under the stereomicroscope to see if the holes contain drilling dust. If dust is present, use tungsten needles to remove it.
8. To sterilize the holder, place it in a conical tube full of distilled water and sonicate it for 20 min with a minimum 20 W power output.

2. Media preparation

1. To heat-inactivate the complement system, leave two 500 μL aliquots of commercial or homemade²⁰ rat serum at 56 °C for 30 min.
   NOTE: For a detailed protocol on how to prepare rat serum, see²¹.
2. In a cell culture hood, prepare two aliquots of the medium for culturing the embryos in the microscope. For each, mix 490 μL of DMEM for live-cell imaging, 500 μL of inactivated rat serum, and 10 μL of penicillin-streptomycin to obtain a final concentration of 50 µg/mL penicillin and 50 µg/mL streptomycin²².
   NOTE: The volume of culture medium depends on the time and number of embryos to be cultured. As a rule of thumb, optimal growth requires a minimum of 200 μL per embryo per day in E7.0 embryos.
3. Using a 2 mL syringe, filter the medium through a 0.22 µm pore size membrane filter into a new tube and place it lid-open in a cell culture incubator at 37 °C and 7% CO₂ for 1 h before embryo culture²³.
4. Prepare the dissection medium by adding to a 500 mL of DMEM supplemented with L-glutamine bottle the following: 50 mL of fetal bovine serum, 10 mL of 25 mM HEPES-NaOH (pH 7.2), and 10 mL of penicillin and streptomycin (50 mg/mL).
5. Next, separate it into 50 mL aliquots, place three aliquots in a 37 °C bath and store the rest at 4 °C for up to 3 months.

3. Embryo dissection

1. Euthanize an E6.75-E7.5 pregnant mouse.
2. Extract the uterus and place it on a dry and clean paper wipe. Then, cut the uterus, inserting the tip of fine scissors from the mesometrial side and sliding the blade along to expose the deciduae and transfer them to the dissection medium. For a detailed protocol, see²⁴.
3. Dissect the embryos, keeping the ectoplacental cone intact (Figure 3A,B). For a detailed dissection method, refer to²⁵.
4. Then, peel off the Reichert's membrane, leaving part of it on the extraembryonic region (Figure 3B).
   NOTE: Fine forceps are essential for this step. Inexperienced users can dissect embryos on 2% agarose gel-coated dishes to avoid bending the forceps' tips against the dish surface (Figure 3C).
5. To keep the dissection medium warm, replace it every 10 min. Moreover, dissect deciduae in groups of 3-4 while keeping the rest in dissection medium placed in a 37 °C bath. Immediately place the dissected embryos in culture medium.
6. Select the embryos with the desired fluorescence features using a fluorescence stereomicroscope and place them in the tube containing culture medium in the cell culture incubator.
7. If the embryos are at E6.5 to E7.0 stages, close the lid and leave the embryos to recover for 2 h before imaging.
   ​NOTE: E6.5 to E7.0 embryos are sensitive. They can also be placed directly under the microscope but preculturing allows for selection of the ones that best recover from dissection, increasing the chance of success.

4. Microscope preparation

1. Turn on all microscope components, including lasers, computer, and software. Turn on the temperature controllers and set to 37 °C 3 h in advance to ensure equilibration.
2. If using an immersion detection objective, clean it using a residue-free disposable wipe. Dip the objective's tip in double-distilled water using a 60 mm dish and leave it until the acquisition starts.
3. Turn on the CO₂ controller and set it to 7%.
4. Place a drop of high-vacuum silicon grease on a 35 mm dish with glass coverslip bottom (14 mm diameter), place the holder on top of the drop, and press gently to immobilize it.
5. Under a stereomicroscope, add culture medium to fill the glass-bottom portion of the dish. While doing so, aim the stream to the holder's holes to prevent formation of air bubbles in them.
6. If the bubbles remain, use the glass capillary prepared in step 1.2, connected to a silicone tube with a mouthpiece, to suck the bubbles out gently.
   ​NOTE: The elbowed end of the capillary will facilitate access through the holes.

5. Embryo mounting

1. Transfer 2-3 embryos that were left to recover in the incubator to the dish with the holder. Leave the rest in the incubator as a backup.
2. Set the embryos in the holes by using a pair of forceps and a tapered tungsten needle. Use the needle to hook the embryo by the ectoplacental cone and insert it into a size-matching hole. To immobilize the embryo, rotate the cone by 90-120° so that the Reichter's membrane adheres to the hole walls (Figure 3C). For an alternative description, see²⁶.
3. Carefully move the dish containing the embryos and the holder to the microscope plate (Figure 3F,G).
   NOTE: If not moved steadily, the embryo can move out of the holder.
4. Lower the objective until a liquid meniscus is formed at the medium-objective interface.
5. Locate the embryo using transmitted light under the microscope binocular and place the embryo in focus.
6. Mount the incubation chamber and seal it around the objective using tape (Figure 3F).
7. Using a Live Capture at the microscope control software, adjust the Laser Output and Gain levels.
   NOTE: For two-photon acquisition of green fluorescent protein (GFP) and Tdtomato reporters, 30% output laser power at 980 nm and 70% gain on non-descanned detectors were used in this protocol (Figure 3G).
8. To avoid evaporation, cover the medium with paraffin oil by dripping it slowly on the objective.
   ​NOTE: This is the last point where additional embryos can be mounted in the holder. Once covered with paraffin oil, one would have to clean the holder and replace the medium to do so. For alternative mounting methods see²⁷.

6. Image acquisition

1. Set up time-lapse recording. Set a 5-10 min time interval with 3-5 µm z-space between stacks. Leave a blank space on top of the z-stack to anticipate embryo growth, a minimum of 50 µm, adding 30 µm for every hour the acquisition will not be supervised.
2. If available, enable the Auto-Save option to allow supervision of the acquisition from a personal computer to allow the adjustment of the z-stack size, if needed, to fit the area of interest within focus.

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.

Representative Results

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.

Discussion

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.

Materials

Name	Company	Catalog Number	Comments
#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