This manuscript describes a technique for visualization of the developing vasculature. Here we utilized in utero intra-cardiac FITC-labeled tomato lectin microinjections on mouse embryos. Using this technique, we delineate the perfused and unperfused vessels throughout the embryonic kidney.
The formation and perfusion of developing renal blood vessels (apart from glomeruli) are greatly understudied. As vasculature develops via angiogenesis (which is the branching off of major vessels) and vasculogenesis (de novo vessel formation), perfusion mapping techniques such as resin casts, in vivo ultrasound imaging, and micro-dissection have been limited in demonstrating the intimate relationships between these two processes and developing renal structures within the embryo. Here, we describe the procedure of in utero intra-cardiac ultrasound-guided FITC-labeled tomato lectin microinjections on mouse embryos to gauge the ontogeny of renal perfusion. Tomato lectin (TL) was perfused throughout the embryo and kidneys harvested. Tissues were co-stained for various kidney structures including: nephron progenitors, nephron structures, ureteric epithelium, and vasculature. Starting at E13.5 large caliber vessels were perfused, however peripheral vessels remained unperfused. By E15.5 and E17.5, small peripheral vessels as well as glomeruli started to become perfused. This experimental technique is critical for studying the role of vasculature and blood flow during embryonic development.
During embryonic development two discrete, yet simultaneous, vascular processes take place: angiogenesis, the process whereby a vessel grows from a major pre-existing vessel, and vasculogenesis, which is a de novo formation of vessels from residential endothelial progenitors1,2. Respectively, the former is synonymous with blood flow, while the latter is thought to largely take place in the absence of it.
Simultaneous to blood vessel formation, a cyclical and dynamic process of kidney progenitor cell synthesis, proliferation, and differentiation begins to unfold on embryonic day 9.5 (E9.5). At this point the ureteric bud (UB) invades dorsally into surrounding metanephric mesenchyme (MM), and continues until birth3. Repeated branching of the UB into rapidly condensing metanephric cap mesenchyme begins the formation of the functional units of the kidney, the nephron. With every new generation of UB and nephron, older generations are displaced into inner cortical and medullary regions, where they then undergo further maturation and differentiation within primarily vascular-dense environments. As evidenced by Dressler et al.3, this embryological process is precipitated by inductive signaling, such as crosstalk between UB and MM, and a myriad of extracellular factors 3-6. Two recently investigated extracellular factors within the developing pancreas and kidneys include oxygen tension and blood flow7,8. The latter will be discussed in further detail below with relation to kidney development.
In order to expose the inductive role that blood flow potentially plays in nephron progenitor cell differentiation, as well as in other organogenesis processes, precise and accurate methods of embryonic blood flow mapping is imperative.
Alternative methods of gauging blood flow include the prescription of ultrasound imaging and resin casts9,10. Conclusively, these modes have been shown to be inherently lacking in their capacity to contemporaneously unveil temporal and spatial juxtapositions between blood flow and stem cell differentiation. Resin casts, for example, provide a valid model of vessel patterning within adult tissues, however in immature vessels, such as with embryonic time points, vessels are grossly underdeveloped and leaky. Therefore, resin casts fail to hold within the tiny, oftentimes porous, vessels.
For these apparent obstacles, among others, we chose to incorporate ultrasound-guided in vivo intra-cardiac embryonic tomato lectin (TL) microinjections into our investigations of kidney development. In this procedure we utilize an ultrasound probe to synchronously guide a mounted micropipette needle filled with 2.5 μl of TL solution into the left ventricle of mouse embryos at E11.5, E13.5, E15.5, and E17.5 time points. E17.5 is the latest developmental age as the needles are not strong enough to penetrate the more developed embryo.
The advantages of this microinjection method are abundant. Ultrasound-guided microinjection allows precise positioning of an injection needle within the embryonic left ventricle, passive and controlled expulsion of solution into the beating heart of the animal, minimal damage to heart and surrounding tissues, and the avoidance of sudden cardiac failure and death of the embryo prior to full-body perfusion. With the use of a FITC-labeled TL, any perfused vasculature will maintain the marker along its endothelial apical membrane. In combination with immunohistochemistry, utilizing Pecam (CD31, Platelet endothelial cell adhesion molecule) and various other vascular markers, we are able to clearly distinguish between perfused and un-perfused vessels, as well as characterize any aberrant staining of surrounding tissues.
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NOTE: The University of Pittsburgh Institutional Animal Care and Use Committee approved all experiments.
1. Preparation of Ultrasound-microinjection Instruments and Embryos
- Set up stage, mount, and probe (Figure 1) as well as surgical instruments (Figure 2). Place phosphate buffered saline (PBS) solution (pH7.4) in a 37 °C warming bath. Fill microinjection needle entirely with mineral oil, using 1 ml syringe attached to a second flexible 25 G needle, through its base.
- Fix needle onto rotation mount arm, and empty needle of mineral oil solution. Re-fill with 2.5 μl of TL solution. Ensure that no air bubbles are present within the injection needle. Rotate needle arm toward wall, away from the stage.
- Anesthetize the pregnant mother in anesthesia chamber via continuous infusion of isoflurane. When mother is rendered unconscious, transfer the anesthesia to nose tube positioned on the caudal side of the stage, and place mother in supine position with snout in nose tube to allow a continuation of a fully anesthetized state.
- Tape limbs in 45° angles, or with hands and feet rested and positioned overtop ECG/Temperature monitor tabs. It is important that the pregnant dam is continually monitored to ensure that anesthesia is sufficient and that ointment in applied to the eyes to reduce drying.
- Apply hair removal product across lower abdomen of the mother, gently wipe off with dry cotton swabs, and then again with a 70% ethanol-saturated cotton swabs to remove any excess product and hair. Ensure to clean abdominal skin off of all hair at site of incision (Figure 3).
- Perform a laparotomy on the pregnant mother using fine surgical forceps and scissors. Take care to avoid cutting any major vessels or visceral organs. Make first, straight incision of epidermis 1.5-2 cm above vagina and continue cut towards ribs for approximately 2-3 cm. Expose the subcutaneous membrane, locate the linea alba (“white line”) (Figure 3), and cut parallel to initial incision, along the same length, to expose internal visceral organs and uterine saccules.
2. Extraction of Embryos
- Use 6-inch cotton-tipped applicators to maneuver mother and embryos. Gently push (do not force) on skin with applicator to manipulate first embryo out of incision opening. After extraction of the first embryo saccule, gently and slowly pull the remainder of the uterine horn through and onto the mother’s exterior. Avoid pulling intestine or other organs through incision at this stage.
- Quantify embryos and gently place left uterine horn back into mother. Then begin placing the right uterine horn back into mother starting with the embryo saccule furthest from the vagina on the horn and moving downward. Continue this until only two embryos remain exposed (Figure 4). Lastly, position embryos in a column above and parallel to incision line, being cognizant not to cut off uterine circulation.
- Place clay blocks and position between arms and body, as well as legs and tail. Ensure that the clay surfaces are approximately level with laparotomy incision. Wet the fenestrated Petri dish with 37 °C PBS.
- Grasp extending forceps with right hand and fenestrated Petri dish with left hand (fenestration parallel to embryos) and bring closed forceps ~5-cm through fenestration, from the top of the Petri dish. Open forceps completely without tearing fenestration mesh.
- Maneuver hands above embryos and focus sight on the two embryo saccules. Without (or lightly) touching saccules with fenestration meshing or forceps, slide them through slit and set Petri dish onto mother’s abdomen. With forceps still extended, manipulate fenestrated mesh to seat it on either side and at base of each embryo (Figure 5) and pull forceps out of slit opening.
- Next, place blue rubber containing wall in Petri dish, without pinching or injuring embryos (Figure 6). Make sure clay blocks are firmly balancing Petri dish, embryo, and rubber-containing wall. Lastly, fill Petri dish with 37 °C PBS until embryos are completely submerged (Figure 6), while controlling for leaks at fenestrated meshing.
3. Injection Procedure
- Lower injection stage with mother and embryos down using Z-level adjustment knob (Figure 1) and then rotate injection needle and mount arm and line up directly with ultrasound probe, with needle ½ cm to the left and under the probe tip (Figure 7). Push needle-arm (not needle) 45° away from the probe. Be careful not to hit the tip of the needle with the dish or ultrasound probe.
- Raise injection stage back to original height, with ultrasound probe directly above embryos Probe must be slightly submerged and within 3-4 mm of the embryonic tissue. Use X & Y stage adjustment knobs (Figure 8) to amend embryo/mother/stage position to systematically locate the beating embryonic heart.
- Directly Center of ultrasound screen observe a black center target marker drawn (*). Locate left ventricle (preferable) or atrium using the X & Y stage adjustment knobs (Figure 8) and then raise or lower the stage using revolving knob on the stage to position injection target precisely over the black center marker on the ultrasound screen.
- Use X stage adjustment knob to move embryo to the right, off of the ultrasound screen. Reposition microinjection needle and arm back into place, ½ cm away from and 90° to the ultrasound probe (Figure 7).
- Using microinjection mount adjustment knobs (Figure 9C-G), position needle with the tip clearly aligned with the center marker. Adjust injection angle (using knobs in Figure 9C & E-G), and X, Y, and Z vectors of micro-needle to ensure needle tip is focused in the correct plane. Retract microinjection needle using solely the “injection” knob (Figure 9F), careful not to adjust other dimensions.
- Again, using solely the X fine-adjustment stage knob move embryo back under the probe, with target exactly on the center mark on the ultrasound screen. Ensure that the left ventricle is now exactly on the same X, Y, and Z plane.
- Next, with just the “injection” knob on the microinjection mount, slowly puncture embryo with the microinjection needle and gradually bring tip into the left ventricle. Inject the full 2.5 μl of TL solution into left ventricle or until the empty warning beep sounds, by holding “empty” and tapping “fill” once (Figures 2A & B). At this time of injection observe a shadow emanating from the tip of the needle which is the TL entering into the cardiac chamber (Figure 10). In reverse, quickly retract microinjection needle rotating the “injection” knob (Figure 9F) on the microinjection mount when injection is complete.
- Continue on to next embryo and repeat this procedure for remaining embryos following this series of steps and finally place the final embryos back into the dam’s abdomen. Switch anesthesia to chamber box and gently move mother here for 15 min from time of the last injection.
4. Harvesting Embryos and Analysis
- Sacrifice dam via cervical dislocation. Expand the laparotomy incision to easily remove uterine horns from the mother. Keep the embryos within uterine sac. Place embryos in chilled PBS.
- Dissect embryos from uterine sac (if necessary collect tissue for genotyping) and place the whole embryo in 4% Paraformaldehyde (PFA) overnight, then into 30% sucrose overnight, freeze in cryostat sectioning medium. Then cut the cryosection and place it onto slides for immunohistochemical analysis. Optionally, use tissues for whole-mount by dehydrating in 100% methanol, following fixation in 4% PFA.
- For immunohistochemical analysis place the cryosections into PBS to remove the OCT. Then block the sections with 10% normal donkey serum for 30 min. Then add PECAM primary antibody at 1:100 dilution overnight at 4 °C. The next day, wash the slides in PBT 3 times for 10 min each and add donkey anti rat 594 secondary and incubate for 1 hr at RT.
NOTE: This is a highly demanding technique requiring the optimization and coordination of ultrasound and microinjection. There is a very steep learning curve related to this technique and requires four to six weeks of mentored injections before one becomes proficient.
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Vascular formation precedes flow in developing kidney
A majority of embryonic tissue (including the kidney) contains a dense vasculature (both unperfused and perfused), even at early embryonic time points. To better gauge and analyze blood flow within the developing kidney we utilized a method of in utero embryonic intracardiac microinjections. With the use of a high-resolution ultrasound to identify the embryonic heart at E11.5 through E17.5, and following the extraction and exposure of a single uterine saccule via a laparotomy, we were able to inject 2.5 μl of TL (fluorescein isothiocyanate (FITC)-conjugated).
We have found the greatest success injecting E13.5 and E15.5 embryonic time points, with a success rate of approximately ~80% to 90%, respectively. Because of the presence of a relatively immature heart at E11.5 and an existence of dense cartilage and formation of bone at E17.5, the outlying time points are comparatively difficult to inject, with a substantially lower probability of success in comparison to the middle embryonic time points. One may expect at least ~50% and ~30% success rates with E11.5 and E17.5 time points, respectively.
By co-labeling tissue with Pecam (CD31), a universal endothelial marker, we are able to qualitatively categorize fluorescent vessels and tissues into three groups: perfused vasculature (Pecam positive & TL positive), unperfused vasculature (Pecam positive), and aberrantly perfused structures (TL positive). This particular staining pattern allows us to spatially distinguish between areas of perfusion and un-perfusion (Figure 11).
At E11.5, we discovered that perfused vessels encapsulate the developing kidney in a web-like pattern without actually penetrating into the organ (Figure 12B). By E13.5, major vessels were perfused with a few of the smaller accessory vessels staining with TL (Figure 12C). Staining could also be seen throughout some of the ureteric epithelium, this may either be from glomeruli that are already perfused or due to the inherent leakiness of early embryonic vessels. By E15.5 major vessels were perfused, however a large number of glomeruli could also be observed holding the TL stain, suggesting that significant filtering is occurring (Figure 12D). Also at this time point a number of smaller caliber vessels appear perfused, although a significant proportion of the outer nephrogenic zone appears to be devoid of blood flow. Lastly, however, by E17.5 a majority of the kidney was perfused except for vasculature of the nephrogenic zone, which is predominately devoid of perfusion (however dense in Pecam positive vessels) thus indicating an absence of angiogenesis in peripheral regions. Smaller caliber vessels contain TL and the average number of perfused glomeruli has dramatically increased in comparison to earlier time points (Figure 12E).
Figure 1. Ultrasound probe, surgical stage, microinjection system, rail system, and ECG/Temperature monitor basic set up.
Figure 2. Necessary surgical equipment, solutions, and devices. (A) Cotton-tipped applicators. (B) Extending (Ring) forceps. (C) Surgical scissors. (D) Blunt-tipped forceps. (E) Fine Forceps. (F) Microinjection Needle (Origio, #C060609). (H) Injection/Fill controller. (I) Phosphate Buffered Saline (PBS). (J) Mineral Oil (Sigma Aldrich).
Figure 3. A 2-cm laparotomy is performed on anesthetized mother, exposing the subcutaneous layer (abdominal wall) above the uterus. Utilizing the surgical scissors and fine forceps, expose the linea alba and make incision parallel to epidermal cut. Please click here to view a larger version of the figure.
Figure 4. 1-2 E15.5 uterine saccules extracted through laparotomy incision and exposed. Please click here to view a larger version of the figure.
Figure 5. Exposed uterine saccules guided through slit in fenestrated Petri dish using extending forceps. Fenestrated meshing seats snuggly at base of the saccules.
Figure 6. Blue containing wall placed to the right of uterine saccules. Petri dish filled with 37 °C PBS until embryos and containing wall are completely submerged. Clay blocks placed securely under Petri-dish/embryo between arms and body and legs and tail.
Figure 7. Injection system positioned ½ cm to the left and below, and 90° to ultrasound probe.
Figure 8. Stage X and Y adjustment mechanisms.
Figure 9. Empty/Fill device, microinjection system, and stage mount. (A) Fill button. (B) Inject button. (C) Circular needle angle adjustment knob. (E) Fine X adjustment knob. (F) “Injection” knob. (G) Course X adjustment knob. (H) Stage revolving height-adjustment knob.
Figure 10. Ultrasound image of E15.5 intra-cardiac TL microinjection. Needle tip is positioned within cardiac chamber, TL solution is indicated by black shadow within two chambers. Pericardium and pericardial cavity are easily differentiated.
Figure 11. Areas between perfusion, unperfusion, and aberrant staining discerned. The first panel shows the Pecam stain. The middle panel displays the TL stain over exactly the same area of tissue. Ureteric bud is aberrantly stained; major vessels are encased by white dotted lines. The last panel is the merge. White arrows indicate vessel leakage, and gray arrows demonstrate a transition towards a vessel becoming perfused. Yellow structures represent fully perfused vasculature. Please click here to view a larger version of the figure.
Figure 12. Embryo and kidney perfusion ontogeny is investigated. (A) Whole E11.5 embryo is perfused, with the very peripheral vessels within head and tail regions holding the TL. (B) Perfused vessels were seen surrounding but not incorporated into the developing kidney at E11.5 in a web-like pattern, there is also quite a large amount of diffuse staining at this point. (C) E13.5 kidney shows major, large caliber vessels are perfused throughout kidney (white and yellow arrow), with perfusion absent within nephrogenic zone (white dotted line). (D) E15.5 kidney shows perfusion throughout small caliber, peripheral vessels (yellow arrows), in some glomeruli (white arrows), and again an absence of perfusion at peripheral kidney mantel. (E) E17.5 kidney shows vast perfusion throughout glomeruli (white arrows) and small caliber vessels (yellow arrows). The nephrogenic zone is indistinguishable from the rest of the kidney at this magnification. Please click here to view a larger version of the figure.
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Microinjection anesthesia and time frame
With regards to anesthetization of the mother, it is essential to keep airflow constant (2-3 L/min) and at low PSI. The flow of the sedative must be held at approximately 1.75-2 L/min. Simultaneously, timeframes in which the injections take place must be closely monitored and controlled for with each litter. For each litter the injection procedure should be kept under 45 min. The importance of this time limit is paramount to the experiment, as each embryo must be maintained within a constant, normal heart rate range in order to facilitate controlled and invariable perfusion throughout the body and organ of interest. We have found that lengthening injections results in dubious results and fluctuations in perfusion patterns between embryos and lowers rates of successful injections (i.e., slow heart rate, cardiac death). Lastly, after the final injection within the litter, it is essential to allow approximately 15 min before embryos are harvested to facilitate proper, full perfusion within each embryo.
Procedural treatment and care of embryos
During extraction of the embryos, we have found greater rates of success with injections that are accomplished by constantly attending to the overall health and integrity of the uterine lining and embryos during the embryo extraction. Using the delicate cotton-tipped applicators (saturated with PBS) to manipulate and maneuver the uterine sac and saccules are imperative to ensure the survival of the mother and embryos throughout the procedure. First, it is important to ensure that the physical integrity of the uterus and placenta is properly maintained. Avoid contorting the saccules in a manner that will obstruct blood flow to the embryos or cause significant breakages in blood vessels that supply blood flow to the placenta. This can be accomplished by extracting a maximum of 1-2 embryos at a time (unless extracting the entire uterine sac for initial count) and by positioning applicators away from major blood vessels at the base of the placenta.
Location of the Embryonic Injection Site
Guiding the microinjection needle in the proper location with the ultrasound machine is also critical for each injection attempt. Careful attention must be given to the locating and centering of the left ventricle on the center marker. Following this, the needle must be located and centered precisely on the center marker as well to align the needle and injection site on the same X and Y plan. Once this is complete, slowly draw the needle tip closer to the injection site and avoid applying undue stress to the pericardial sac and heart. This is done by allowing the needle to gradually pass through tissue layers, rather than forcing the passage of the needle through them. During the injection, it is also important to look out for a black shadow emanating from the needle tip (Figure 10). This is indicative of the TL solution filling the ventricle. If the needle tip is in the correct position, the shadow should continuously appear and disappear as TL is ejected into the aorta. However, If the pericardial cavity seems to engorge, this is indicative that the needle is off target of the left ventricle and the TL solution is filling the pericardial cavity surrounding the heart. This is a common mistake that is easily corrected by repositioning the injection needle on its X and Y plane, with any slight adjustments taking place with the needle still within the embryo. Avoid extracting the needle from the embryo at this stage.
Ultrasound-guided in vivo microinjection limitations
Inherently, the ultrasound microinjection possesses a number of fundamental technical limitations. First and foremost, the microinjection needle volume capacity restricts the age range of injections to E17.5 in the mouse. 2.5 μl of TL solution has been shown in our studies to completely perfuse embryos up to E15.5. However, just following this time point, the TL to blood volume ratio becomes to minuscule and diluted to effectively tag peripheral renal endothelial membranes. Thus, a diminished picture of blood flow is present at later time point injections. Additionally, the formation of bone and cartilage in the embryo creates a natural barrier for the needle tip, thus leading to lengthened injection times and frequent cracks in the needle head. These limitations may be overcome by utilizing a novel injections system with greater needle strength and volume capacities.
Alternative embryonic blood flow mapping techniques
To date, alternative methods of mapping embryonic mouse blood flow include the implementation of resin casts, immunohistochemistry staining, and high resolution ultrasound imaging. As resin casts (with the integration of advanced imaging techniques) are currently the most popular alternative, this will be discussed in further detail below. Resin casts allow for the creation of accurate three dimensional representations of flow within postnatal organs. However, little success has been had with imaging prenatal renal tissue due to porous blood vasculature and limitations in resolution. In comparison, conjugated TL stains enable bonding to endothelial membrane antigens leading to a higher degree of precision. In other cases, resin cast viscosity prematurely truncates flow into glomeruli vessels and tiny capillary beds, creating investigational limitation at higher magnifications. At lower magnifications, this technique is clearly viable at establishing flow patterns in relation to developing regions. Conversely, TL allows high-resolution analysis of blood flow relative to developmental structures at the cellular level.
Future application and directions
Our data suggests that blood flow and oxygenation are critical factors with regards to nephron progenitor differentiation. To further elucidate their roles in renal development, future investigations must delineate underlying anatomical mechanisms precipitating this physiological process as well as examine transcriptional signaling pathways that mediate this phenomenon as well. One hypothesis, with regards to bridging the gap between phenomenon and mechanism, is that smooth muscle cell and pericyte aggregates play contributory roles in orchestrating the transient truncation of blood flow into stem cell regions of developing organs. In order to test this, further immunofluorescent staining and analysis must be conducted with a focus on these cell types at the nephrogenic zone border. In terms of future investigations of underlying signaling pathways, we would like to explore the roles of hypoxia inducible factors and the Von Hippen Lindau signaling pathways. Both of these have largely been implicated throughout literature as playing important inductive roles in maintaining hypoxic and oxygenated environments within stem cell regions (largely seen to be hypoxic) and areas of differentiation (areas in greatest need of oxygenation), respectively. Furthermore, we would like to interrogate the role of erythropoietin (EPO) in mediating the processes of angiogenesis and vasculogenesis throughout kidney vascular development. EPO is a renal glycoprotein that plays critical roles in endothelial differentiation and maintenance. Its role in blood mediated endothelial differentiation is largely unknown. Lastly, we would like to conduct in vivo microinjection experiments with vasodilation compounds to further strengthen the validity of these investigations.
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The authors declare that they have no competing financial interests.
The authors would like to thank Dr. George Gittes for advice and expertise throughout this study. SSL was supported by an American Heart Association fellowship (11POST7330002). Further to this SSL and this study was supported by an NIDDK Mentored Research Scientist Development Award (DK096996) and by the Children’s Hospital of Pittsburgh.
|DAPI||Sigma Aldrich||022M4004V||concentration 1:5,000|
|Pecam||BD Biosciences||553370||concentration 1:100|
|FITC-Tomato Lectin||Vector Laboratories||FL-1321||concentration 2.5 µl / embryo|
|Alexa Fluor-594 (Donkey Anti-Rat)||Jackson Immunoresearch||712-585-150||concentration 1:200|
|Microinjection Needle||Origio Mid Atlantic Devices||C060609|
|Mineral Oil||Fisher Scientific||BP26291|
|1 ml syringe||Fisher Scientific||03-377-20|
|Clay Blocks||Fisher Scientific||HR4-326|
|Surgical Tape||Fisher Scientific||18-999-380|
|Hair Removal Product||Fisher Scientific||NC0132811|
|Surgical Scissors||Fine Science tools||14084-08|
|Fine Forceps||Fine Science tools||11064-07|
|Surgical Marking Pen||Fine Science tools||18000-30|
|Right angle forceps (for hysterectomy)||Fine Science tools||11151-10|
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