Immunohistochemical labeling of blood vessels with isolectin B4 in whole mounts of embryonic telencephalon:
Whole mount preparations described below were key in identifying fine anatomical details and developmental gradients of telencephalic vasculature (Fig. 2). By using these methods, we found that the periventricular vessels in the ventral telencephalon originated from a prominent basal vessel located deep on the floor of the telencephalic vesicle in the basal ganglia primordium (Fig. 2a). The basal vessel unfurled into a plexus and during the E9 to E11 interval grew in a ventral to dorsal and lateral to medial direction, straddling the lateral ventricles (Fig. 2e-i). The periventricular plexus was restricted to a ~20 µm-thick plane parallel to the pial surface and sandwiched between the ventricular and marginal zones in the E11 dorsal telencephalon (Fig. 2b-d). Narrow branches from the periventricular network joined the pial plexuses as fine, tapering vessels. The specific steps in the preparation of the whole mounts are as follows.
1| Collect mouse embryos one by one from deeply anesthetized dams (Ketamine, 50 mg/kg and Xylazine, 10 mg/kg; i.p.).
2| Decapitate each embryo immediately upon removal from the mother and remove the brains.
3| Immerse the brains in 4% paraformaldehyde in 0.1M phosphate buffer, pH 7.2 and store at 4°C. For whole mounts from E15 or older embryos, the brain should be removed from the skull prior to fixation.
4| Prior to preparation of the whole mounts, wash the brains three times in phosphate buffered saline (PBS; 0.1M phosphate buffer, pH7.3 and 0.9% Sodium Chloride) and permeabilize with 1% bovine serum albumen (BSA) and 0.5% TritonX-100 in PBS at 4oC overnight.
5| Following a PBS rinse for 5 min, incubate the brains in biotinylated isolectin B4 (1:40, Sigma) in antibody diluent solution (BD Pharmingen) containing 1% TritonX-100 at 4oC overnight.
6| After six washes in PBS (each wash for 4 min), incubate the brains with Alexa 594 streptavidin conjugate (Invitrogen) diluted 1:200 in antibody diluent (BD Pharmingen) containing 0.5% TritonX-100 for 6 hours at 4oC. Rinse brains six times in PBS, 4 min for each rinse.
7| Place the brains in a 35 mm Petri dish containing PBS and view under a dissecting microscope (Fig. 3a).
8| Separate the telencephalon from the rest of the brain (Fig. 3b) by severing the nascent internal capsule as close to the ganglionic eminences as possible using spring scissors (2-4 mm blade length).
9| Separate the telencephalic hemispheres from each other by a mid-line cut using a scalpel blade (number 11; Fig. 3c). Cut each hemisphere along the caudal to rostral direction and open like a book (Fig. 3d) to reveal the ganglionic eminences at the base of the telencephalon.
10| Mount the open hemisphere flat with the ventricular surface facing the viewer (Fig. 3e) on Superfrost Plus glass slides with Vectashield hard setTM mounting medium (Vector Laboratories) and examine using a Zeiss Pascal LSM 5 laser confocal microscope.
Endothelial cell migration in explants of embryonic mouse telencephalon in culture:
We found that the periventricular vessels develop in a ventral-to-dorsal and lateral-to-medial gradient. The periventricular vessel developmental gradient is established as a result of migration of endothelial cells from the ventral to the dorsal telencephalon beginning around E10. To verify periventricular endothelial cell migration across telencephalic compartmental boundaries, heterochronic explantation studies using explants of the E11 ventral telencephalon and E10 dorsal telencephalon are used (Fig. 4). Since the endothelial cells have not yet entered the dorsal telencephalon in vivo at E10, use of the E10 dorsal telencephalon explants in the heterochronic explantation studies confers a unique advantage because any endothelial cell that is found in the E10 dorsal telencephalon explant can be assumed with certainty to have originated in and migrated from the ventral explant.
1| Collect embryos by hysterotomy of deeply anesthetized dams and decapitate.
2| Place the embryonic brains in sterile petridishes in ice cold NeurobasalTM medium (Gibco) that contains 2% B27 supplement and Penicillin-streptomycin-Glutamine mix (Gibco).
3| Under a microscope, cut the telencephalon along the caudal to rostral direction with fine microtip scissors. Trim the floor plate gently to flatten the tissue.
4| Separate the dorsal and ventral telencephalon by making an incision at the dorso-ventral boundary.
5| Transfer each telencephalic explant to polycarbonate membranes (8 mm pore size; Gibco) in sterile 6 well plates containing NeurobasalTM medium. Place the plates in the incubator (370C, 5% CO2 and 95% humidity) and take out as and when required.
6| Test cell viability in the explants with a LIVE/DEAD® Viability/Cytotoxicity Kit (Invitrogen). We observed cell death only in regions that were mechanically manipulated during micro-dissection (Fig. 4a). Perform all procedures under sterile conditions.
7| For pre-explantation labeling with QDot® nanocrystals, incubate the explant with Qtracker reagents (Qtracker 655 Cell Labeling Kit; Invitrogen) in NeurobasalTM medium for 45 min at 37oC. The nanocrystals provide stable intense fluorescence that is not lost over time. The nanocrystals are useful as pre-explantation markers for the entire explant rather than for labeling individual endothelial cells within the explant.
8| Ventral explants from Tie2-GFP mice are a better option as endothelial cells express GFP and can be monitored during their migration into the dorsal explant.
9| Transfer the dorsal explant to the polycarbonate membrane and graft the labeled ventral explant onto the unlabeled dorsal explant using fine tungsten needles. During this process, visualize the labeled explant with a fluorescence microscope (Nikon Eclipse E400) to confirm appropriate positioning (Fig. 4b, e).
10| Incubate the explants for 24 h at 370C (5% CO2 and 95% humidity) in NeurobasalTM medium.
11| At the end of the culture period, detach the explants from the membrane filters gently into petridishes and fix in zinc fixative (BD Pharmingen) at room temperature for 24 h. The zinc fixative is a milder fixative and helps preserve many antigenic epitopes producing superior labeling with many antibodies when compared to paraformaldehyde fixation.
12| Transfer the explants into a glass vial and dehydrate it in ascending series of ethanol (1 h each in 70% and 95% ethanol and two changes of 1 h each in 100% ethanol) and clear in xylene (1h, twice). During this process place the vial in a Rotamix. Glass vials are preferred because plastic vials may not withstand the xylene exposure well. Next, fill the glass vial (containing the explants) to 25% capacity with fresh xylene and transfer to a 60oC oven. Following 15 min, add molten paraffin wax (Peel-away embedding paraffin; Polysciences) to the vial so that the ratio of wax to xylene is 1:1. After 30 min, add more paraffin to bring the ratio of xylene to paraffin to 1:2 and 30 min later replace the paraffin-xylene mixture with fresh paraffin. Following overnight immersion in the fresh paraffin at 60oC embed the explants flat in paraffin in a plastic mould (Peel-away embedding molds; Polysciences) and leave at room temperature for 24 h. Remove the paraffin block containing the explant from the mold, trim it and section on a rotary microtome in the coronal plane at 15 µm thickness. Mount the sections on Superfrost Plus glass slides (Fisher Scientific) and dry at 37oC for 24 h. Deparaffinize the sections in xylene (two changes, 10 min each), rehydrate in descending series of ethanol (100%, 95%, 70%, 10 min each) and transfer to PBS. Paraffin processing provides high quality histology for the delicate explants when compared to cryo-preservation techniques. The sections are ready for immunohistochemistry with various markers (Figure 4c, d, f). Sections are pre-treated with BD Retrievagen (pH 6.0) (BD Pharmingen) before immunohistochemical staining by using standard methods.
Transplantation of mouse brain derived endothelial cells into explants of telencephalon
To study endothelial cell autonomous role of ventral and dorsal transcription factors in telencephalic angiogenesis, we knocked down the transcription factor genes in cultured embryonic mouse endothelial cells by using siRNA technology and transplanted the cells into E11 wild type CD1 ventral telencephalon explants to study their migratory behavior. Embryonic mouse endothelial cells prepared from Nkx2.1-/- and SeyDey mice were also used in parallel studies. Here we describe transplantation of siRNA transfected E13 mouse brain derived endothelial cells into E11 ventral telencephalic explants.
To prepare primary endothelial cell cultures from embryonic mouse ventral telencephalon, collect E13 embryos, dissect the telencephalon and separate its ventral and dorsal compartments by an incision at the dorso-ventral boundary. Mince tissue pieces into 1-2 mm fragments with a scalpel blade, rinse in PBS and incubate at 37oC for 0.5 h in pre-warmed PBS with trypsin (0.25%), DNase I (1 mg/ml) and EDTA (0.5mM). Dissociate the tissue, spin it down, re-suspend in warm 10% fetal calf serum (FCS)-DMEM and filter through a sterile 40 µm nylon mesh. Collect cells, resuspend in 1 ml RBC lysing buffer (Sigma), overlay onto DMEM and centrifuge briefly. Resuspend the cell pellet in 1 ml of 10% FCS-DMEM.
Coat dynabeads with sheep anti-rat IgG (Invitrogen) and incubate with rat anti-mouse CD31 (PECAM-1) monoclonal antibody (BD Pharmingen) at 4oC overnight and wash three times with 2% FCS in PBS. Add 1 ml cell suspension to the tube containing the washed beads. After 30 min at 4oC with occasional agitation, recover bead-bound cells using a magnetic field, wash five times in 10% FCS-DMEM and once with FCS-free DMEM and then digest for 10 min at 37oC in 1 ml of trypsin/EDTA (GIBCO) to release the beads. Centrifuge bead-free cells in 10% FCS-DMEM and re-suspend in endothelial cell culture medium (BIOCOATÒ Endothelial cell growth environment, BD Biosciences). Culture cells in 75 cm2 BIOCOATÒ Collagen Type 1 flasks (BD Biosciences) and feed every 2 days with complete medium exchange and trypsinize for subculture at 80% confluency. Split cells into 6 well plates and maintain in DMEM before transfection with siRNA constructs.
Use pre-designed siRNA constructs for Nkx2.1 (ON-TARGETplus SMARTpool, L-041979-01, Dharmacon) and a control non-targeting siRNA (D-001206-14-05, Dharmacon). Co-transfect a siGLO Red transfection indicator (Dharmacon) with siRNA to serve as an indicator for transfection success. Achieve optimal conditions by transfecting endothelial cells at 80% confluency in DMEM in 6 well plates; transfect with siRNA (2 µM) and siGLO Red transfection indicator (1 µM), using DharmaFECT transfection reagent 1; and follow manufacturer’s protocols. Observe virtually complete knockdown of NKX2.1 under such conditions, within 96h by western blotting and immunocytochemistry. Before the transplantion step, spin down cells and collect in 200 ml DMEM. These endothelial cells are now ready for transplantation into explants.
Prepare explants of E11 mouse CD1 telencephalon as described earlier and place on polycarbonate membrane (8 mm pore size; Gibco) in sterile 6 well plates in DMEM.
For ideal transplantation of endothelial cells, make sure that the explant is not fully submerged in the medium. The upper surface of the explant should be exposed to air so that the surface tension will help the transplanted cells adhere to the explant.
For the transplantation procedure itself, take approximately 100 endothelial cells in a fine micropipette and pressure inject focally into the explant. Leave the plates untouched for 15-20 minutes and then transfer into the incubator. One hour after the transplantation, add more medium to the wells to fully submerge the explants.
After 24 hours, process the explants for paraffin wax histology and immunohistochemistry. The siGLO Red transfection indicator is resistant to the paraffin processing steps and is an excellent tool to identify transplanted endothelial cells. Control-transfected endothelial cells migrate from the site of transplantation into the host ventral explant in 24 h (Fig. 5a-c). On the other hand, virtually all the Nkx2.1 siRNA transfected endothelial cells are restricted to the site of transplantation (Fig. 5d-g).
Histological sections or whole mounts of the embryonic mouse telencephalon are ideal for discerning and quantitatively illustrating gradients of blood vessel development following immunohistochemical labeling of the vessel components. The whole mounts are especially valuable because they permit a global view of the entire vascular network while preserving its anatomical relationships. Explants of the dorsal and ventral telencephalon maintained in culture are ideal for illustrating endothelial cell migration across telencephalic compartments. Although manipulation of the neural tube or embryonic telencephalon for preparation of the explants is a delicate procedure requiring micro-dissection expertise, the technique is a valuable tool for studying a variety of cellular and molecular mechanisms in developmental biology. Combining the explant culture, cell transplantation and siRNA technologies permitted us to study the role of specific genes in the regulation of cross-compartmental migration of endothelial cells.