Erratum to: The importance of microglia in the development of the vasculature in the central nervous system
Vascular Cell. 2013;
Received: 10 June 2013 | Accepted: 10 June 2013 | Published: 25 June 2013
Vascular Cell ISSN: 2045-824X
After the publication of this work  it was brought to our attention that citations in the article were not correspondingly numbered in the reference list. To avoid confusion, the article is republished here in its entirety, with the citations referenced correctly.
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The body’s vascular system is thought to have developed in order to supply oxygen and nutrients to cells beyond the reach of simple diffusion. Hence, relative hypoxia in the growing central nervous system (CNS) is a major driving force for the ingression and refinement of the complex vascular bed that serves it. However, even before the establishment of this CNS vascular system, CNS-specific macrophages (microglia) migrate into the brain. Recent studies in mice point to the fundamental importance of microglia in shaping CNS vasculature during development, and re-shaping these vessels during pathological insults. In this review, we discuss the origin of CNS microglia and their localization within the brain based on data obtained in mice. We then review evidence supporting a functional role of these microglia in developmental angiogenesis. Although pathologic processes such as CNS ischemia may subvert the developmental functions of microglia/macrophages with significant effects on brain neo-angiogenesis, we have left this topic to other recent reviews [2, 3].
Microglia – specialized macrophages of the CNS
Microglia are specialized macrophages of the central nervous system involved in immune regulation, tissue development, homeostasis and wound repair. Microglia were first observed by Virchow in the mid-nineteenth century (see ), and described in greater detail by Pio del Rio-Hortega in 1932. In this almost prescient work, del Rio-Hortega described microglia morphology, plasticity during development and with pathological insult, their cellular origin, and microglia association with white matter tracts and blood vessels. Despite an immense amount of research on microglia origin and function since then, these early views remain surprisingly accurate.
Microglia derive from primitive yolk sac macrophages
Microglia belong to the mononuclear phagocytic system - a family of cells that includes committed precursors in the bone marrow, circulating blood monocytes and tissue macrophages in every organ of the body including the CNS . Mononuclear phagocytes are typified by their ability to ingest large particles; their morphology; their expression of common surface markers including CD11b, CD68, Colony Stimulating Factor 1 Receptor (CSF1R), chemokine receptor CXCR3, and plasma membrane glycoprotein F4/80 ; and their presumed hematopoietic origin . While microglia certainly meet the functional and morphological definition of a mononuclear phagocyte [7–9], their developmental origin has until recently been less clear.
In mice, hematopoietic stem cells (HSCs) emerge from the dorsal aorto-gonado-mesonephros (AGM) region 10.5 days after conception (embryonic day (E) 10.5), then migrate to the fetal liver where they expand and differentiate before definitive hematopoiesis in the spleen and bone marrow [10–13]. In adult mice, blood monocytes, classical dendritic cells, and certain tissue macrophages derive from, and are continuously replaced by, bone marrow-derived HSCs. It was previously thought that microglia arose from hematopoietic precursors in two waves of recruitment and differentiation [14, 15]. However, it is now clear, based on evidence from bird, fish and mammals, that yolk-sac derived macrophage precursors contribute significantly, if not entirely, to the brain’s microglia. In avian embryos, analyses using chick-quail transplantation and parabiosis chimeras show that yolk sac-derived macrophages migrate to and invade the CNS through the pial basal lamina before and independent of CNS vacularization [16, 17]. Subsequent live recordings of cell movements in zebrafish embryos revealed that yolk sac-derived macrophages migrate through the cephalic mesenchyme before its vascularization to reach the brain pial surface and the roof of the 4th ventricle, from where they subsequently invade the neuroepithelium and eventually acquire microglial characteristics . Recently, fate mapping studies in the mouse using genetic lines such as
The yolk sac origin of microglia is further supported by elegant experiments done in mice lacking the transcription factors PU.1 [20, 25–27] or Myb , and in mice with inactivated
Patterns of brain colonization by microglia
Once born, yolk-sac derived macrophage precursors migrate into and colonize the whole (mouse) embryo between E9.5 and E10.5 (Figure 1) [19, 20]. The first organ to be colonized is the brain. Subsequent phases of microglial brain colonization follow a stereotyped pattern (see ). Microglia invade the brain through the pial surface, then migrate and proliferate, populating the brain in a dorsal-to-ventral and rostral-to-caudal gradient. During this time, microglia associate with radial glia and blood vessels, and are found in close proximity to dying cells. Eventually, microglia are notably excluded from the neuroepithelium and cortical plate, and then are widely distributed in the adult brain, except in areas of densely packed neuron cell bodies such as the pyramidal cell layer. The early association of microglia around blood vessels has led to the hypothesis that microglia may enter the brain through the developing vasculature. Indeed, E10.5
Microglia shape the developing CNS vasculature
Because the developing CNS lacks intrinsic vasculature, CNS blood vessel development occurs exclusively via angiogenesis [39–41]. Attracted by proangiogenic signals, new capillaries sprout from perineural vessels, and invade the neuroectoderm around E10 in mice. These nascent capillaries are composed of tip cells at the vascular front, followed by proliferative stalk cells. Tip cells extend filopodia toward guidance cues such as VEGF-A. VEGF-A induces expression of the Notch ligand, Dll4, predominately in tip cells. Dll4 then activates Notch in adjacent cells, which down-regulates VEGF receptors and up-regulates angio-suppressive factors like sFlt1 and Jagged-1, promoting a stalk cell phenotype (reviewed in ). The interplay between VEGF and Notch signaling is highly regulated with additional inputs from other major signaling pathways including BMPs [42, 43], Semaphorins , and Wnt/βcatenin [45, 46]. Additional signaling pathways that regulate tip cell formation and sprouting include sphingosine-1-phosphate and its receptor S1pr1 [47–49]. During vascular sprouting, tip cells anastomose with neighboring tip cells, creating vascular loops. In this way, vessels sprout, extend, branch and anastomose, iteratively, toward the center of the neural tube where they establish a temporary plexus, termed the periventricular vascular plexus (PVP), around the CNS ventricular spaces and spinal cord’s central canal [50–52]. As the CNS grows and differentiates, these vessels associate with microglia, pericytes, neuroepithelial radial glia and neuroblasts, and later astrocytes; CNS vessels are refined, arteries and veins are established, and the mature neurovascular system takes form.
The retina and optic nerve represent highly specialized extensions of the forebrain. While its vascularization occurs by angiogenic sprouting similar to the brain, the timing and scaffolds that guide angiogenesis are partly different . During the first week of life, an astrocytic network arising from the optic nerve invades into the retina in a centrifugal fashion. As the primitive hyaloid (hv) vessels that supported the embryonic eye development regress, a new primary vascular plexus extends into the retina, following structural and morphogenic cues provided by astrocytes and Müller glia. At 7 to 9 days of postnatal life, vessels sprout perpendicularly into deeper layers of the retina, forming a deep vascular plexus in the outer plexiform retinal layer (OPL). During the next 3 weeks, retinal vasculature continues to sprout, remodel and differentiate into arteries and veins and a mature neurovascular network is established by 6 weeks of age.
Microglia influence CNS vascular development
As discussed above, microglia migrate into the CNS and retinal neuroepithelium before vessels do. Microglia are therefore uniquely positioned to influence the early sprouting, migration, anastomosis, and refinement of the growing CNS and retinal vascular systems. Studies of angiogenesis after microglial depletion, or in mice lacking microglia, strongly support this concept. Checchin et al.  administered clodronate liposomes either systemically to deplete macrophages and circulating monocytes, or intravitreally, which depleted retinal microglia without reducing circulating monocytes. These authors found that reducing retinal microglia numbers was associated with a decrease in retinal vascular density. Selective depletion of circulating monocytes had no impact on retinal blood vessels. Importantly, intravitreal co-administration of microglia with clodronate restored vascularity of the developing retina, suggesting a microglia-specific effect on retinal vascular development. Similarly, Kubota et al.  found that
The concept that microglia may act to “bridge” vascular sprouts during CNS vascular development was introduced by the work of Fantin et al. . This group studied vascular ingression and branching in embryonic hindbrains and retinas of mice lacking macrophages and microglia (
A recent report from our laboratory confirmed and extended many of these observations . We found that microglia-deficient mice (
In contrast to these findings, Stefater et al.  recently uncovered a mechanism whereby microglia may
The feedback loop between VEGF and Notch involves regulation of both VEGFR-2 and VEGFR-3, although the individual contribution of each of these VEGF receptors remains unclear [40, 57–59]. The primary ligands for VEGFR-3, VEGF-C and VEGF-D, are highly expressed by microglia, and VEGF-C-positive microglia are found near the fusion points of VEGFR-3-positive vascular sprouts . While
Other groups have recently explored potential roles for Notch signaling in microglia-endothelial cell interactions. Outtz et al.  found that Notch signaling is activated in retinal microglia, which are closely associated with endothelial tip cells expressing the Notch ligand Dll4. Moreover, genetic deletion of Notch1 in retinal microglia led to a subtle reduction in the numbers of microglia found at the vascular front. Interestingly, Hoffman et al.  found that the Notch ligand, Jagged1, is highly expressed in perivascular cells of the retina, including microglia. Further studies should evaluate the specific roles of Notch signaling in microglia, and the impact of this signaling on retinal vascular development.
The development of organ-specific vascular beds is dependent upon the close communication between the vascular cells (endothelial cells and mural cells) on the one hand, and resident cells of the organ, on the other. The CNS vasculature is in many ways unique in its anatomy and regulation, and it harbors a highly specific barrier – the blood–brain, or blood-retina, barrier. The development of the CNS vasculature hence occurs in tight association with the development of other components of the CNS, and as a result of reciprocal communication between the endothelial cells and different emerging CNS cell types. This review focuses on the role of microglial cells – a CNS-specific type of macrophage – and also mentions in passing the importance of other cells types, such as radial glial cells and astrocytes, as sources of VEGFs, Wnts and other signaling molecules that control the shape and function of the emerging vasculature. Although recent studies provide compelling evidence for a role of microglia in shaping the nascent vascular plexuses in the brain and retina, the molecular mechanisms remain to be elucidated, and the possibility remains that microglia play a different role at different locations, for example in the different retinal capillary plexuses. It also remains to be shown what role, if any, microglia play in vascular homoeostasis in adult physiological and pathophysiological processes. Microglial cells become activated in conjunction with pathological insults and disruption of the blood–brain barrier, where they may play protective or pathogenic roles. These data are not discussed in the present review, which is focused on physiological development. However, the awareness of microglia as a unique CNS cell type with a distinct ontogeny and equipped with specific developmental and pathological functions compared to other glial cell types, has recently increased. Our perspectives on these cells will undoubtedly grow rapidly in the coming years.
Animal experiments were approved by the Stockholm’s North Ethical Committee for Animal Research.
This work has been supported by a Leducq Foundation Transatlantic Network of Excellence and a Transatlantic Career Development Award.
Authors’ original submitted files for images
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