Entry, dispersion and differentiation of microglia in the developing central nervous system.

Microglial cells within the developing central nervous system (CNS) originate from mesodermic precursors of hematopoietic lineage that enter the nervous parenchyma from the meninges, ventricular space and/or blood stream. Once in the nervous parenchyma, microglial cells increase in number and disperse throughout the CNS; these cells finally differentiate to become fully ramified microglial cells. In this article we review present knowledge on these phases of microglial development and the factors that probably influence them.

Proliferation of actively migrating ameboid microglia in the developing quail retina.

Sheets containing the inner limiting membrane covered by a carpet of Müller cell endfeet were used to show that ameboid microglial cells migrating tangentially in the vitreal part of the developing retina of quail embryos underwent mitosis. Double labeling with anti-beta-tubulin/QH1 or Hoechst 33342/QH1 revealed that some migroglial cells with morphological features typical of active migration were in early prophase. By anaphase and early telophase, microglial cells had retracted their lamellipodia and were ovoid in shape. Later in telophase, but well before completion of cytokinesis, both daughter cells again emitted lamellipodia, thus regaining the typical morphology of migrating cells. We concluded that ameboid microglial cells go through cycles in which migration and mitosis alternate, and that both mechanisms contribute to the spread of microglia throughout the developing retina. The mitotic spindle of dividing microglial cells showed different orientations, which probably influenced the course of subsequent migration. The expression of the proliferating cell nuclear antigen in the nucleus of most tangentially migrating ameboid microglial cells at E9-E10 confirmed their proliferative capability. However, the rate of proliferation of these cells decreased during embryonic development, and was nearly zero at E14.

Circumferential migration of ameboid microglia in the margin of the developing quail retina.

Central-to-peripheral migration of QH1-positive microglial precursors occurs in the vitrealmost part of the developing quail retina. This study shows that some QH1-positive ameboid cells with morphological features of migrating cells are already present in the margin of the retina before microglial precursors migrating centrally to peripherally arrive in this zone. Because the earlier cells are oriented parallel to the ora serrata, we deduce that some microglial cells migrate circumferentially in the margin of the retina, whereas other microglial precursors migrate from central to peripheral zones. Microglial cells that migrate circumferentially are first seen on embryonic day 6 (E6) and advance in a temporal-to-dorsal-to-nasal direction from the temporoventral quadrant of the retina. When cells migrating centrally to peripherally reach the retinal margin, they meet those migrating circumferentially. From E6 on, some QH1-positive dendritic cells in the ciliary body bear processes that penetrate the retina, where they are oriented circumferentially. These observations suggest that microglial cells that migrate circumferentially in the retinal margin share a common origin with dendritic cells of the ciliary body. Therefore, microglial cells of the quail retina appear to make up a heterogeneous population, with some cells originating from the pecten/optic nerve head area and others from the ciliary body.

Naturally occurring cell death and migration of microglial precursors in the quail retina during normal development.

We compared chronotopographical patterns of distribution of naturally occurring neuronal death in the ganglion cell layer (GCL) and the inner nuclear layer (INL) with patterns of tangential and radial migration of microglial precursors during quail retinal development. Apoptotic cells were identified by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling technique, and microglial precursors were identified by immunocytochemistry with an antibody recognizing quail microglial cells (QH1 antibody). Apoptotic cells were first detectable in the GCL at the seventh day of incubation (E7), were most abundant at E10, and were absent after E13. In the INL, apoptotic cells first appeared at E7, were most abundant at E12, and disappeared entirely after the third posthatching day (P3). In both retinal layers, cell death first appeared in a small central area of the retina and subsequently spread along three gradients: central-to-peripheral, temporal-to-nasal, and dorsal-to-ventral. The chronology of tangential (between E7 and E16) and radial migration (between E8 and P3) of microglial precursors was highly coincident with that of cell death in the GCL and INL. Comparison of the chronotopographical pattern of distribution of apoptotic nuclei in the GCL with the patterns of tangential and radial migration of microglial precursors neither supported nor refuted the hypothesis that ganglion cell death is the stimulus that triggers the entry and migration of microglial precursors in the developing retina. However, microglial cells in most of the retina traversed the INL only after cell death had ceased in this layer, suggesting that cell death in the INL does not attract microglial precursors migrating radially. Dead cell debris in this layer was phagocytosed by Müller cells, whereas migrating microglial cells were seen phagocytosing apoptotic bodies in the nerve fiber layer and GCL but not in the INL.

Tangential migration of ameboid microglia in the developing quail retina: mechanism of migration and migratory behavior.

Long distance migration of microglial precursors within the central nervous system is essential for microglial colonization of the nervous parenchyma. We studied morphological features of ameboid microglial cells migrating tangentially in the developing quail retina to shed light on the mechanism of migration and migratory behavior of microglial precursors. Many microglial precursors remained attached on retinal sheets containing the inner limiting membrane covered by a carpet of Müller cell endfeet. This demonstrates that most ameboid microglial cells migrate tangentially on Müller cell endfeet. Many of these cells showed a central-to-peripheral polarized morphology, with extensive lamellipodia spreading through grooves flanked by Müller cell radial processes, to which they were frequently anchored. Low protuberances from the vitreal face of microglial precursors were firmly attached to the subjacent basal lamina, which was accessible through gaps in the carpet of Müller cell endfeet. These results suggest a mechanism of migration involving polarized extension of lamellipodia at the leading edge of the cell, strong cell-to-substrate attachment, translocation of the cell body forward, and retraction of the rear of the cell. Other ameboid cells were multipolar, with lamellipodial projections radiating in all directions from the cell body, suggesting that microglial precursors explore the surrounding environment to orient their movement. Central-to-peripheral migration of microglial precursors in the retina does not follow a straight path; instead, these cells perform forward, backward, and sideways movements, as suggested by the occurrence of (a) V-shaped bipolar ameboid cells with their vertex pointing toward either the center or the periphery of the retina, and (b) threadlike processes projecting from either the periphery-facing edge or the center-facing edge of ameboid microglial cells.

Microglia development in the quail cerebellum.

We used the QH1 antibody to study changes in the morphological features and distribution of microglial cells throughout development in the quail cerebellum. Few microglial precursors were present in the cerebellar anlage before the ninth incubation day (E9), whereas many precursors apparently entered the cerebellum from the meninges in the basal region of the cerebellar peduncles between E9 and E16. From this point of entry into the nervous parenchyma, they spread through the cerebellar white matter, forming a 'stream' of labeled cells that could be seen until hatching (E16). The number of microglial cells in the cerebellar cortex increased during the last days of embryonic life and first posthatching week, whereas microglial density within the white matter decreased after hatching. As a consequence, the differences in microglial cell density observed in the cerebellar cortex and the white matter during embryonic life diminished after hatching, and microglia showed a nearly homogeneous pattern of distribution in adult cerebella. Ameboid and poorly ramified microglial cells were found in developing stages, whereas only mature microglia appeared in adult cerebella. Our observations suggest that microglial precursors enter the cerebellar anlage mainly by traversing the pial surface at the basal region of the peduncles, then migrate along the white matter, and finally move radially to the different cortical layers. Differentiation occurs after the microglial cells have reached their final position. In other brain regions the development of microglia follows similar stages, suggesting that these steps are general rules of microglial development in the central nervous system.

Macrophages during avian optic nerve development: relationship to cell death and differentiation into microglia.

Cell death is frequent during the development of the nervous system. In the developing optic nerve of chicks and quails, neuroepithelial cell death was first observable on the third day of incubation, slightly after the first cell ganglion axons appeared in the stalk. Specialized phagocytes were observed within the stalk in chronological and topographical coincidence with cell death. These cells were identified as macrophages because of their morphological features, intense acid phosphatase activity and, in quail embryos, labeling with QH1, a monoclonal antibody recognizing quail hemangioblastic cells. Macrophages in areas of cell death were round and actively phagocytosed cell debris. We used electron microscopy and histochemical and immunocytochemical labeling to study macrophagic cells of the optic nerve in avian embryos of 3-6.5 days of incubation. As development proceeded, phagocytosing, round macrophages became ameboid macrophages that migrated from areas of cell death toward regions occupied by optic axonal fascicles. Macrophages in these locations were thin and elongated, with a few processes. To elucidate the final fate of macrophagic cells in the optic nerve, sections taken from older embryonic and hatched quails were stained with the QH1 antibody. On the 8th day of incubation some slightly ramified QH1+ cells were present among axonal fascicles. In subsequent stages these cells increased in number and acquired more complex ramifications. In adult optic nerves, QH1+ cells had a small body and sent out slender processes, sometimes with secondary and tertiary branches, which were frequently orientated parallel to the course of the optic axons. These cells were considered to be microglial cells. The appearance of macrophages within the developing optic nerve at the same time as neuroepithelial cell death suggests that cell death influences the recruitment of macrophages into the nerve. When macrophages reach the areas invaded by optic axonal fascicles, they undergo structural and probably also physiological changes that appear to signal differentiation into microglia.

Origin of microglia in the quail retina: central-to-peripheral and vitreal-to-scleral migration of microglial precursors during development.

The origin, migration, and differentiation of microglial precursors in the avascular quail retina during embryonic and posthatching development were examined in this study. Microglial precursors and developing microglia were immunocytochemically labeled with QH1 antibody in retinal whole mounts and sections. The retina was free of QH1+ macrophages at embryonic day 5 (E5). Ameboid QH1+ macrophages from the pecten entered the retina from E7 on. These macrophages spread from central to peripheral areas in the retina by migrating on the endfeet of the Müller cells and reached the periphery of the retina at E12. While earlier macrophages were migrating along the inner limiting membrane, other macrophages continued to enter the retina from the pecten until hatching (E16). From E9 on, macrophages were seen to colonize progressively more scleral retinal layers as development advanced. Macrophages first appeared in the ganglion cell layer at E9, in the inner plexiform layer at E12, and in the outer plexiform layer at E14. Therefore, it seems that macrophages first migrated tangentially along the inner retinal surface and then migrated from vitreal to scleral levels to gain access to the plexiform layers, where they differentiated into ramified microglia. Macrophages appeared to differentiate shortly after arrival in the plexiform layers, as poorly ramified QH1+ cells were seen as early as E12 in the inner plexiform layer and at E14 in the outer plexiform layer. Radial migration of macrophages toward the outer plexiform layer continued until posthatching day 3, after which retinal microglia showed an adult distribution pattern. We also observed numerous vitreal macrophages intimately adhered to the surface of the pecten during embryonic development, when macrophages migrated into the retina. These vitreal macrophages were not seen from hatching onwards, when no further macrophages entered the retina.