In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 progenitors^1,2. 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 flow^7,8. 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 casts^9,10. 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.

Protokół

NOTE: The University of Pittsburgh Institutional Animal Care and Use Committee approved all experiments.

1. Preparation of Ultrasound-microinjection Instruments and Embryos

1. 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.
2. 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.
3. 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.
4. 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.
5. 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).
6. 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

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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

1. 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.
2. 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.
3. 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.
4. 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).
5. 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.
6. 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.
7. 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.
8. 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

1. 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.
2. 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.
3. 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.

Wyniki

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 sin...

Dyskusje

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 embr...

Materiały

Name	Company	Catalog Number	Comments
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
PBS	Fisher Scientific	NC9763655
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