Monitoring endothelial cell development and migration in the embryonic CNS

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.