- Methodology article
- Open Access
High resolution ultrasound-guided microinjection for interventional studies of early embryonic and placental development in vivoin mice
© Slevin et al; licensee BioMed Central Ltd. 2006
- Received: 08 August 2005
- Accepted: 27 February 2006
- Published: 27 February 2006
In utero microinjection has proven valuable for exploring the developmental consequences of altering gene expression, and for studying cell lineage or migration during the latter half of embryonic mouse development (from embryonic day 9.5 of gestation (E9.5)). In the current study, we use ultrasound guidance to accurately target microinjections in the conceptus at E6.5–E7.5, which is prior to cardiovascular or placental dependence. This method may be useful for determining the developmental effects of targeted genetic or cellular interventions at critical stages of placentation, gastrulation, axis formation, and neural tube closure.
In 40 MHz ultrasound images at E6.5, the ectoplacental cone region and proamniotic cavity could be visualized. The ectoplacental cone region was successfully targeted with 13.8 nL of a fluorescent bead suspension with few or no beads off-target in 51% of concepti microinjected at E6.5 (28/55 injected). Seventy eight percent of the embryos survived 2 to 12 days post injection (93/119), 73% (41/56) survived to term of which 68% (38/56) survived and appeared normal one week after birth. At E7.5, the amniotic and exocoelomic cavities, and ectoplacental cone region were discernable. Our success at targeting with few or no beads off-target was 90% (36/40) for the ectoplacental cone region and 81% (35/43) for the exocoelomic cavity but tended to be less, 68% (34/50), for the smaller amniotic cavity. At E11.5, beads microinjected at E7.5 into the ectoplacental cone region were found in the placental spongiotrophoblast layer, those injected into the exocoelomic cavity were found on the surface or within the placental labyrinth, and those injected into the amniotic cavity were found on the surface or within the embryo. Following microinjection at E7.5, survival one week after birth was 60% (26/43) when the amniotic cavity was the target and 66% (19/29) when the target was the ectoplacental cone region. The survival rate was similar in sham experiments, 54% (33/61), for which procedures were identical but no microinjection was performed, suggesting that surgery and manipulation of the uterus were the main causes of embryonic death.
Ultrasound-guided microinjection into the ectoplacental cone region at E6.5 or E7.5 and the amniotic cavity at E7.5 was achieved with a 7 day postnatal survival of ≥60%. Target accuracy of these sites and of the exocoelomic cavity at E7.5 was ≥51%. We suggest that this approach may be useful for exploring gene function during early placental and embryonic development.
- Implantation Site
- Pregnant Mouse
- Uterine Horn
- Neural Tube Closure
- Fluorescent Bead
In utero microinjection of mouse embryos has proven valuable for exploring the developmental consequences of altering gene expression using adenoviral or retroviral vectors [1–12], or for injecting cells to study cell lineage or migration [13, 14]. Microinjection is also a useful approach for rescuing mutant embryos to validate gene therapies and/or circumvent embryonic morbidity or mortality thereby permitting study of a gene's role later in development or adulthood . This approach has been largely limited to studying embryos at embryonic day 9.5 (E9.5) or greater when the uterus and decidua have become thinner and the placenta and embryo are relatively large so that ultrasound or trans-illumination can be used to guide injections into the conceptus in the exteriorized uterus. However, knocking out or mutating genes critical for early development of the yolk sac, chorioallantoic placenta, hematopoietic system, embryonic heart and vasculature often cause embryonic lethality before E9.5 [16–18]. Thus, the goal of the current study was to develop and validate methods to accurately target specific regions of the conceptus under high-resolution ultrasound guidance at E6.5 to E7.5 of gestation. At this stage, the embryo is not yet dependent on placental or circulatory function for survival. We show that ultrasound-guided microinjection into the ectoplacental cone region at E6.5 or E7.5 and the amniotic cavity at E7.5 was achieved with a 7 day postnatal survival of ≥60% and that target accuracy of these sites and of the exocoelomic cavity at E7.5 was ≥51%, suggesting this method will provide a feasible approach for future studies.
Ultrasound imaging of the early embryo
At E7.5, the amniotic and exocoelomic cavities, and the ectoplacental cone region were each discernable on ultrasound (figure 1C). When compared to an appropriate histological section at the same gestation (figure 1D), the structural detail in the ultrasound image can be appreciated. As at E6.5, the 3D impression of the E7.5 conceptus observed in real-time (additional file 2) was clearer than in 2D still images (figure 1C)
Dissection to establish conditions for accurate bead microinjection
The maximum injection volume that resulted in few or no beads off-target was determined in 4 mice at E7.5 as follows. We began by microinjecting an aqueous suspension of 3 μm diameter green fluorescent beads into the amniotic cavity at a volume of 207 nL, which was approximately 50% of the lowest previously published volume . This caused marked distension of the cavity and surrounding area so we gradually reduced the volume to 13.8 nL in successive experiments. Injection accuracy and frequency of beads in adjacent off-target areas was visually evaluated using a stereomicroscope in implantation sites that were dissected as previously described  within a few hours following microinjection.
Target accuracy following micro-injection.
Beads in conceptus2
Beads localized in target3
Within 24 hours
Day of injection
Day of injection
Day of injection
Assessment of embryonic survival and effect of development on localization of beads
Survival following microinjection or sham procedures.
age at intervention
Embryo 2–12 d postinjection
95% CI for % alive
95% CI for % alive
1 wk Postnatal
95% CI for % alive
Ectoplacental cone region
Following microinjection into the ectoplacental cone region at E6.5, 78% of embryos in 10 mice were still alive when assessed 2 to 12 days later, 73% of pups from 4 mice were still alive at birth, and 68% were still alive at 1 week of age (table 2). In 7 of the 10 mice evaluated during pregnancy, the conceptus was dissected within 7 days post injection and the localization of beads was assessed by gross morphology. 74% (62/84) of the embryos were alive of which 95% (59/62) had beads in the conceptus. 95% (56/59) of these had beads predominantly localized to the placental region.
We performed sham experiments to assess the effect of microinjection on embryo viability. We also determined survival in non-operated control pregnancies. For sham 1, all experimental procedures at E7.5 were replicated except no microinjection was performed (surgical exposure, ultrasound imaging, uterine manipulation, and experimental duration were replicated). For sham 2, uterine horns were exposed at E7.5 to count the number of implantation sites and then were immediately replaced. For normal survival in non-operated control pregnancies, we determined embryonic number as a function of gestational age in control mice without prior interventions. We examined an average of 15 pregnant mice per gestation day from E6.5 to E18.5 at necropsy, and counted the number of viable embryos. Linear regression was used to show that the average number of embryos at E0 was 14 and the rate of embryo loss per day was 0.13. This information was used to calculate the average embryonic survival rate in pregnancies without interventions at specific gestational ages.
In sham 1, 54% of embryos were alive and appeared normal when assessed at 1 week postnatal age (table 2). This rate did not differ significantly from survival rates following microinjection into the amniotic cavity (60%) or the ectoplacental cone region (66–68%) (i.e. there was overlap in 95% confidence intervals) indicating that the embryonic lethality was predominantly due to the surgical procedure not to the microinjection procedure. In sham 2, survival at 1 week postnatal age was 84% which was significantly higher than in Sham 1 (54%) (table 2). Sham 2 survival at birth (84%) was close to the 89% survival expected for non-operated control pregnancies. Thus, optimal embryonic survival depends upon keeping the duration of the surgical procedure short and minimizing uterine manipulation.
Ultrasound-guided microinjection provides a feasible approach for experimental interventions into either the embryonic (E7.5) or extra-embryonic (E6.5–7.5) regions. Limitations include the presence of beads in off-target locations and a significant increase in embryonic deaths. In the current study we injected fluorescently labeled microspheres in vehicle (0.9% saline). In future studies the vehicle could include inhibitory RNA, transfection vectors, specific antibodies, or labeled cells and thus this method could be used to explore the developmental consequences of altering gene expression, or for studying cell lineage and migration. However, whether the localization of cells, small molecules or solutes would be similar to that of the beads used in the current study is unknown. This limitation may be addressed by microinjecting bioactive agents tagged or absorbed to spheres, or by microinjecting cells that are labeled so that spheres or cells can be localized later either grossly during dissection or in histological sections.
We speculate that microinjections into the exocoelomic cavity may be useful for studies of the development of the yolk sac (e.g. vascularization and hematopoiesis), the interactions between the allantois and chorioallantoic placenta (e.g. chorio-allantoic fusion and morphogenesis) and the development of the placental labyrinth layer. In addition, the ectoplacental cone region can be targeted directly, thereby placing agents among relatively undifferentiated, early placental trophoblasts. Since umbilical blood flow does not commence until approximately E9.5 [20, 25], injected agents are likely to remain within this target location and not circulate systemically in the embryo unlike placental injections performed in later gestation. Microinjections into the amniotic cavity may be useful for influencing early events in embryonic development such as gastrulation, axis formation, and neural tube closure.
The study protocol was approved by the Mount Sinai Hospital Animal Care Committee, and was conducted in accord with the guidelines of the Canadian Council on Animal Care.
Animals and animal-related procedures
A modified Petri dish (100 mm diameter, 25 mm deep), similar to that used in earlier work on older embryos [1, 2, 13, 14, 26, 27], was positioned above the incision site as follows (figure 8C). The Petri dish had a 25 mm hole in the bottom, which was sealed from below by a thin transparent rubber membrane (40 mm square ). The membrane had a 10 × 1 mm slit cut in the centre, which was positioned above the skin incision (figure 8C). Fine forceps were passed through the slit in the rubber membrane, the skin edges were gently apposed, and pulled up while the dish and membrane were placed against the maternal skin. Pulling up on the skin in this manner helped create a watertight seal as well as keeping the skin accessible so it could later be held in place while retrieving and replacing the uterus. Using fine forceps, the slit edge and its apposing skin edge were lifted to reveal the underlying uterine segment. Using a second pair of forceps, the uterine segment was gently exteriorized by grasping between implantations sites. At this stage, the transparent membrane was checked to ensure it was still adherent to the maternal skin suggesting an intact watertight seal.
The Petri dish was then supported by Plasticene blocks and filled with calcium and magnesium-free phosphate-buffered saline (PBS) (figure 8C). The PBS served as an ultrasound-coupling medium, maintained hydration of the uterus, and was used at room temperature to reduce normal resting myometrial activity (and therefore the associated gross movements of the implantation sites). The exposed uterine segment was short, ~1–3 implantation sites, which helped stabilize the segment during uterine activity and during microinjections. It was supported laterally by a semi-circular sheet of Silicone rubber (Silastic L RTV silicone rubber; Dow Corning ), which was submerged next to it inside the Petri dish on the side opposite that of the microinjector (figure 8C). This 'uterine stabilizer' was cast in a modified Petri dish and then bisected prior to use.
After microinjecting all sites as described in detail below, the maternal abdomen was closed using 6-0 silk, (Sofsilk™, United States Surgical Corporation ) using a continuous suture for the peritoneum and abdominal muscles and interrupted mattress sutures (3–4) for the skin. Anaesthesia was discontinued and the mouse was placed in a heated recovery chamber. Usually the mouse was awake and active within 5–10 minutes. No pregnant mice died or aborted following microinjection at E6.5 or E7.5 in over 100 procedures performed to date.
High frequency ultrasound
We used an ultrasound biomicroscope (UBM) with a 40 MHz probe (Vevo-660™; VisualSonics) for real-time (34 frames/s) microvisualization of the conceptus and microinjection pipette. The 40 MHz probe has a spatial resolution of ~50 μm at 40 MHz . The mouse was mounted on the stage of a rail system (VisualSonics), which permitted the operator to readily adjust the position of the mouse while maintaining the microinjection pipette within the transducer-imaging plane (figure 8A).
Microinjection pipettes were prepared using methods adapted from prior work . Glass microcapillary tubes (3 1/2 "replacement tubes" Drummond Scientific Company, cat. # 3-000-203-G/X ) were pulled using a vertical puller (Narishige PB-7 ) to produce a long taper. Pipettes were broken using forceps or a scalpel blade in the narrowed region under a dissection microscope and the tip diameters were measured using the calibrated length scale in the eyepiece of the microforge (Narishige MF-9). Pipette tips with an outer diameter of approximately 50–80 μm and inner diameter of approximately 40–50 μm were gradually beveled to 20 degrees (figure 8B) using a continuously moistened grinder for 20 minutes (Narishige EG-4). Too little moisture resulted in a high incidence of tips blocked by particles following the grinding process. All tips were carefully inspected after sharpening to ensure the tip was clear and sharp (figure 8B).
The microinjection pipette was filled with mineral oil before connecting it to the microinjector using the manufacturer's instructions (Nanoject II; Drummond Scientific Co.). Once attached, excess oil was expelled. Green fluorescent beads (polystyrene latex, 3.0 ± 0.1 μm diameter, 2.5% solids in water; 1.68 × 103 particles/nl; Fluoresbrite™ YG Microspheres; Polysciences ) were diluted 2:1 in saline. With the aid of a stereomicroscope (Leica, MS5 ) and micromanipulator, the tip of the microinjection pipette was positioned in a 15 μL drop of the bead mixture and the mixture aspirated using the microinjector controls immediately prior to use. The filling procedure was visually monitored using the stereomicroscope to ensure no air was aspirated and that the pipette tip did not touch the bottom of the dish to prevent the tip from being blunted or broken. When the microinjection pipette was filled, the microinjector was transferred to the micromanipulator on the rail system, for alignment within the ultrasound scan plane.
The microinjection pipette was imaged in PBS prior to the experiment. It was aligned in the scan plane using the XYZ controls so that the tip was centred within the focal zone of the transducer (area of image with the greatest resolution). This position, 'the guide point', was recorded by the UBM and remained displayed on the monitor. The pipette was then retracted leaving the tip in PBS to prevent dehydration-induced obstruction of the lumen.
After injecting the 1–3 exposed sites, they were replaced and the uterine segment containing the next 1–3 implantation sites was carefully removed from the abdominal cavity, pulled gently through the water seal membrane into the PBS-filled Petri dish and was laid next to the uterine stabilizer. The patency of the microinjection pipette was confirmed before continuing.
We microinjected the targeted region within each site in both uterine horns (usually 10 to 16 sites) within 20 to 30 minutes (demonstration in additional files 3, 4, 5) using two operators. One operator loaded and discharged the needle and took notes, while the other performed surgery, operated the UBM, and positioned the microinjection pipette in the target area. Abnormal sites that appeared to contain embryos undergoing re-absorption had their positions noted but were not injected.
At necropsy, the localization of beads was visually evaluated using a stereomicroscope (Leica, MS5 ) with an attached universal light source (MAA-002 from BLS Ltd. ) in implantation sites that were dissected as previously described .
Histology was performed on implantation sites that were immersion-fixed in 4% paraformaldehyde, dehydrated in alcohol, and paraffin embedded. Tissue was sectioned (5 μm) and stained with hematoxylin and eosin (H&E) to show general morphology of the implantation site for comparison with ultrasound images (figure 1B, D). In order to detect the fluorescent beads, 50 μm frozen sections of paraformaldehyde-fixed sites were imaged using an inverted microscope with a FITC filter to detect the fluorescent beads. Images showing the beads were superimposed on autofluorescent images of the tissue obtained using a CY-3 filter (tissue autofluoresces red/orange, figure 4) or using immunofluorescence to detect collagen 4 (basement membranes of blood vessels appear pink, figure 5B–D) and/or following counterstaining with 4', 6-Diamidino-2-phenylindole (DAPI; DNA fluoresces blue, figure 6B–D).
95% confidence intervals were used to determine statistical significance when comparing target accuracy between the different target sites at E6.5 and E7.5 and when comparing survival between the experimental and sham groups. Linear regression was used to calculate the average number of embryos at a given gestation in non-operated control pregnancies. P < 0.05 was considered significant.
We would like to thank the following for their advice, guidance and help; Dr. Daniel Turnbull and members of his laboratory for their advice and initial guidance on UBM guided microinjection techniques, and Qiang Xu (histology core Samuel Lunenfeld Research Institute of Mount Sinai Hospital, Toronto) and Dr. Robin N. Han (Terrence Donnelly Heart Centre, Division of Cardiology, St Michael's Hospital, Toronto) for advice and help in preparation of histology slides. We also thank the VisualSonics Company for technical support in apparatus design and construction.
This work was funded by operating grants from the Canadian Institutes of Health Research and equipment funds from the Canadian Foundation for Innovation and the Richard Ivey Foundation. We also thank the Department of Obstetrics & Gynecology of Mount Sinai Hospital, Toronto, Canada and Rotunda Hospital, Dublin, Ireland for scholarship support for JS. SLA acknowledges that she is a member of the Scientific Advisory Board of the VisualSonics Company but otherwise has no financial interest in the company.
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