scientificprotocols authored about 8 years ago
Authors: Anju Vasudevan & Pradeep G. Bhide
The anatomy of the brain’s vascular networks is just as complex as that of its neuronal networks. Yet, surprisingly little is known about the ontogeny of cerebral vasculature. Until now, it was believed that brain’s vascular networks developed passively to meet metabolic needs of the rapidly growing nervous tissue (ref.1,2). Although classical studies identified a ventral to dorsal temporal developmental angiogenesis gradient in the telencephalon (ref.3), the sequence of angiogenesis was considered to merely shadow neurogenesis and neuronal maturation. According to current models (ref.4,5) brain vasculature develops in four stages (Fig. 1a), responding to and keeping pace with the rapid onset and progression of neuroepithelial progenitor cell divisions, neurogenesis and gliogenesis. According to this model, blood vessels on the pial surface extend radial branches towards the ventricle (ventriculo-petal branches; stage 1); form new branches upon arrival in the periventricular region (stage 2); reverse direction to grow back to the pia (ventriculo-fugal branches; stage 3); and finally branch into plexuses (stage 4). This model does not support distinct developmental schedules for pial and periventricular vessels nor the role of transcription factors Nkx2.1, Dlx1, Dlx2 or Pax6 in the development of periventricular vessels (Vasudevan et al 2008). We have proposed an alternative model that can support our recent findings (Vasudevan et al 2008) and also fit with the notion of VEGF-guided vessel growth. In this model (Fig. 1b), pial and periventricular vessels develop according to independent schedules. The pial vessels encompass the embryonic telencephalon as early as embryonic day 9 (E9) and do not display developmental gradients. The periventricular vessels, which form the bulk of the telencephalic vasculature, arise as branches of the basal vessel located in the basal ganglia primordium (stage 1). The periventricular vessel branches form an orderly lattice in the ventral telencephalon (stage 2). Later, the periventricular vessel network propagates into the dorsal telencephalon (stage 3) as a result of migration of endothelial cells, which is controlled by homeobox transcription factors. Thus, a ventral-to-dorsal and lateral-to-medial gradient of telencephalic angiogenesis is established (stage 4). Based on earlier reports (ref.6) we have proposed that the pial vessels may develop into venous sinuses and the periventricular vessels into the arterial network. We arrived at this model of brain angiogenesis based on data collected by multiple techniques including analysis of the distribution of blood vessels and endothelial cells by immunohistochemistry in histological sections and whole mounts of the telencephalon. Using these anatomical methods we also demonstrated that endothelial cells express compartment-specific transcription factors. The other techniques used included explant cultures that were employed to demonstrate that endothelial cells migrated from ventral to dorsal telencephalon. By using telencephalic explants from mouse models with mutations in specific transcription factor genes, we established that ventral transcription factors Nkx2.1, Dlx1 and Dlx2 were required for migration of endothelial cells from ventral to dorsal telencephalon and that the dorsal transcription factor Pax6 was required for migration of endothelial cells within the dorsal telencephalon. In addition we demonstrated cell autonomous effects of homeobox genes on endothelial cell migration by using small interfering RNA (siRNA) in primary cultures of mouse brain endothelial cells to knock down the homeobox transcription factor gene expression.
Animals. Timed pregnant mice are used. The day of vaginal plug discovery is considered embryonic day 0 (E0). All experiments using laboratory animals are approved by the animal care and use committees of Massachusetts General Hospital and conform to NIH guidelines for the care and use of laboratory animals.
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.
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.
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.
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.
Figure 1: A new model of telencephalic angiogenesis
Current model of CNS angiogenesis [a; modified from reference 4] proposes that pial vessels extend radial branches towards the ventricle (ventriculo-petal branches; stage 1); form new branches upon arrival in the periventricular region (stage 2); reverse direction to grow back to the pia (ventriculo-fugal branches; stage 3); and finally branch into plexuses (stage 4). We propose an alternative model (b) in which, pial and periventricular blood vessels develop along independent schedules. The periventricular vessels are branches of the basal vessel located in the basal ganglia primordium (stage 1). The basal vessel produces an orderly lattice of periventricular branches in the ventral telencephalon (stage 2). The periventricular vessel network propagates into the dorsal telencephalon (stage 3) to produce the dorsal periventricular plexus (stage 4), controlled by ventral and dorsal homeobox transcription factors. A larger, pdf version of this figure can be found here.
Figure 2: Angiogenesis gradients in the embryonic telencephalon
(a) Isolectin-B4+ pial vessels covering a E10 dorsal telencephalon, cut open (at arrowheads) and mounted with the ventricular surface up. The basal vessel appears on the telencephalic floor (thick arrow). The dorsal telencephalon is unlabeled (thin arrow). (b–d) Isolectin B4–labeled periventricular vessels in an E11 whole mount appear in a single 20-mm focal plane (b) from which thin vessels emerge at right angles toward the pial surface (c) and contact the pial vessels (arrow in d), which appear in a different focal plane (d). (e,f) Isolectin B4–labeled prominent basal vessel (white star) in E11 ventral telencephalon whole mount unfurls into a periventricular vessel lattice. The broken line (f) indicates the advancing vessel front: the medial telencephalon has no periventricular vessels (white arrow). (g-i) Diagramatic representation of periventricular vessel development. The periventricular vessel network (depicted in red) originates from the basal vessel (red asterisk in g) in the telencephalon (peacock green) and grows in ventral-to-dorsal and lateral-to-medial directions. Dotted circle in g is expanded in h (cartoon in purple) for a two-dimensional view of the periventricular network (yellow dotted circle) and the basal vessel (red asterisk in h). The boxed area in h represents f, with the medial aspects of the telencephalon devoid of periventricular vessels. (i) Ventral-to-dorsal and lateral-to-medial gradients of periventricular angiogenesis (broken red line with directional arrow). Blue dotted line, pial vessels. Scale bars: a, 100 mm (applies to a, e); b, 50 mm (applies to b–d, f). A larger, pdf version of this figure can be found here.
Figure 3: Preparation of embryonic telencephalon mouse whole mounts
(a) The embryonic brain viewed under a dissecting microscope. (b) Each hemisphere is cut along the caudal to rostral direction and opened like a book. (c) The telencephalic hemispheres are separated from each other (d) The ganglionic eminences at the base of the telencephalon is revealed. (e) The open hemisphere is mounted flat with the ventricular surface facing the viewer. A larger, pdf version of this figure can be found here.
Figure 4: Co-culture of mouse telencephalon explants
Figure 5: Migration of transplanted endothelial cells on a ventral explant
(a-c) Control siRNA transfected (a-c) endothelial cells transplanted into E11 CD1 wild type ventral telencephalon and double-labeled with isolectin B4 (a) and SigloRed (b) migrated from the transplantation site (white arrow in a, c) into the explant (yellow arrows). (c) a and b merged. Virtually all of the Nkx2.1 siRNA transfected endothelial cells, identified by isolectin B4 (d) and SigloRed (e) labeling, were restricted to the site of transplantation (white arrow, d-f). (f) d and e merged. (g) Number of Nkx2.1 siRNA treated endothelial cells that migrated was expressed as a percentage relative to that of control siRNA treated endothelial cells. Nkx2.1 siRNA treated endothelial cells were found very close to the transplantation site whereas endothelial cells transfected with control non-targeting constructs migrated far into the explant (t-test; *P<0.0001). A larger, pdf version of this figure can be found here.
Compartment-specific transcription factors orchestrate angiogenesis gradients in the embryonic brain, Anju Vasudevan, Jason E Long, James E Crandall, John L R Rubenstein, and Pradeep G Bhide, Nature Neuroscience 11 (4) 429 - 439 16/03/2008 doi:10.1038/nn2074
Anju Vasudevan & Pradeep G. Bhide, Neurology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA.
Source: Protocol Exchange (2008) doi:10.1038/nprot.2008.82. Originally published online 31 March 2008.