High resolution ultrasound-guided microinjection for interventional studies of early embryonic and placental development in vivo in mice

Abstract

Background: 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.

Results: 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.

Conclusion: 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 > or =51%. We suggest that this approach may be useful for exploring gene function during early placental and embryonic development.

Figure 1

Anatomical detail visible in ultrasound images. Ultrasound images (A, C) and H&E histological sections (B, D) of implantation sites at E6.5 (A, B) and E7.5 (C, D). Divisions in the scale in A and C are 100 μm apart. The conceptus in histological sections is smaller than in vivo due to shrinkage during tissue preparation (fixation and dehydration). AC, amniotic cavity; Al, allantois; Emb, embryo; EPC, ectoplacental cone region; Exo, exocoelomic cavity.

Figure 2

Off-target beads following 69 nL microinjection into the amniotic cavity. This E7.5 conceptus was dissected a few hours after ultrasound-guided microinjection of a 69 nL volume containing 3 μm diameter fluorescent beads into the amniotic cavity. (A) The amniotic and yolk sac cavities were visibly distended (e.g. compare to figure 3D) when viewed under a dissection microscope. (B) When the same embryo was viewed under a fluorescent microscope, beads were visible within the amniotic cavity (arrow) but were also present in the adjacent exocoelomic cavity as well as in the ectoplacental cone region. AC, amniotic cavity; EPC, ectoplacental cone region; Exo, exocoelomic cavity; YSC, yolk sac cavity.

Figure 3

Target accuracy. Concepti dissected a few hours after ultrasound-guided microinjections of a 13.8 nL volume containing 3 μm diameter fluorescent beads. In these examples, there were few (e.g. arrows in A&D) or no beads considered off-target (e.g. B&C). The ectoplacental cone region was targeted in (A) at E6.5 and (B) at E7.5. At E7.5, the exocoelomic cavity (C) and amniotic cavity (D) were also targeted. AC, amniotic cavity; EPC, ectoplacental cone region; Exo, exocoelomic cavity.

Figure 4

Histological detection of injected beads. Implantation site (tissue autofluoresces orange) containing green fluorescent beads collected a few hours following ultrasound-guided microinjection into the ectoplacental cone region at E7.5. Beads visualized in 50 μm frozen sections were primarily localized to the targeted ectoplacental cone region. AC, amniotic cavity; Emb, embryo; EPC, ectoplacental cone; Exo, exocoelomic cavity; YSC, yolk sac cavity.

Figure 5

Localization of fluorescent beads in the spongiotrophoblast layer following ectoplacental cone microinjection. (A) Placement of the micropipette tip (arrow) near the center of the ectoplacental cone region (EPC) at E7.5. The EPC is demarcated by an echogenic 'V'-shape (arrowhead) [19], and is therefore easy to identify on ultrasound. (B-D) Histological images obtained from a conceptus at E11.5 following microinjection of green fluorescent beads into the EPC at E7.5. Frozen sections were counterstained with DAPI (nuclei stain blue) and immunofluorescence was used to detect collagen 4 in the basement membrane of the labyrinth capillaries (pink). The beads are localized in the placental spongiotrophoblast layer between the labyrinth and the decidua. Boxed regions in (B) and (C) are shown as higher power images in (C) and (D) respectively. AC, amniotic cavity; Emb, embryo; Sp, spongiotrophoblast layer; US, uterine stabilizer.

Figure 6

Localization of fluorescent beads in the placental labyrinth following exocoelomic microinjection. Images of placentas dissected at E11.5 following ultrasound-guided microinjection of green fluorescent beads into the exocoelomic cavity at E7.5. (A) Stereomicroscopic image showing fluorescent beads embedded in the fetal surface of the placenta near the cord insertion. (B) 50 μm frozen section through the placenta and implantation site showing fluorescent beads distributed within the labyrinth layer, extending as deep as the border between the labyrinth and spongiotrophoblast layers (dotted line). Boxed regions in (B) and (C) are shown as higher power images in (C) and (D) respectively. Cell nuclei were stained with DAPI in B, C, and D. AC, amniotic cavity; Emb, embryo.

Figure 7

Embryonic localization of fluorescent beads following amniotic cavity microinjection. E9.5 embryo dissected 2 days after ultrasound-guided microinjection of 13.8 nL of fluorescent beads into the amniotic cavity. Green fluorescent beads were visible within the neural system (arrows) as well as on the skin surface. The embryo was imaged using a Leica MZ 16FA stereomicroscope with GFP filter.

Figure 8

Microinjector apparatus and experiment set-up. (A) The rail system maintains the alignment of the image plane of the ultrasound transducer (1) with the trajectory of the microinjector (2). The stage (3) on which the mouse is placed is adjusted to place the target region of the conceptus in the image plane by adjusting the position of the stage using the XYZ controls (4). The rail system increases efficiency by reducing time to target acquisition, improving accuracy and as a result contributes to improved overall survival. (B) The sharp bevelled tip of the microinjection pipette is shown. (C) A modified Petri dish containing PBS (5) is supported above the mouse using Plasticene blocks (6). A segment of the uterus is exposed through a small midline abdominal incision into the Petri dish. A thin transparent rubber membrane (7) attached to the under surface of the Petri dish (5) seals the dish to the dry denuded maternal skin (8), while a thicker silastic membrane (9) submerged within the Petri dish stabilizes the uterus during microinjection.

Figure 9

Practical points to improve accuracy. (A) Diagram of the optimum experimental set-up enlarged from that shown in figure 8. The uterine stabilizer (1) is cast in one of the modified Petri dishes so that it extends into the central hole (2). As a result the uterine stabilizer fits more closely to the transparent membrane (3) attached to the under surface of the petri dish and also creates a more secure seal with the skin of the mouse abdomen (4). This is important because it reduces the possibility of the exposed segment of uterine horn slipping between the under surface of the stabilizer and the transparent rubber membrane or the maternal skin during microinjections. Keeping the exposed uterus closely approximated to the uterine stabilizer (dotted oval labelled 5 in A &C) is very important in improving accuracy of microinjections. This improves stability of the uterine segment during microinjection allowing easier penetration of the micropipette through the thick uterine muscle enabling a more accurate placement of the tip of the microinjection pipette into the target region. (B) If the uterus is separated from the edge of the uterine stabilizer (double-headed arrow), the uterus will move away from the advancing needle and the target area of the conceptus will move out of the focal zone and/or field of view of the transducer reducing the accuracy of the microinjections. (C) The optimal position of the uterus relative to the uterine stabilizer is demonstrated. The alignment of the conceptus within the scan plane is also optimal. In this position three potential targets are easily accessible from a lateral approach thereby avoiding injury to the embryo caused by the microinjection pipette or the inadvertent deposition of fluorescent beads in other targets during needle insertion or withdrawal. (D) Placement of the microinjection pipette within the amniotic cavity (arrow). (E) Immediately after removal of the microinjection pipette, a small amount of echogenic material (fluorescent beads) can be seen within the amniotic cavity (arrow) which was not present prior to microinjection (compare with amniotic cavity in image B).