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.

The development of microglia in the quail optic tectum from embryonic day 6 to adulthood was studied by using the QH1 monoclonal antibody. In youngest tecta, microglial cells were scarcely present, but their number rose in subsequent stages. A clear pattern of microglial cell distribution was observable in embryos of 9-16 days. (1) Round cells appeared close to the ventricular layer. (2) Large numbers of ameboid and round labeled cells were seen in the stratum album centrale during development. A gradient of cell density was observable in this layer, as fewer labeled cells appeared in medial regions of the tectum than in lateral regions. (3) Maturing ramified cells were found in layers external to the stratum album centrale, where they increased in number and in branching complexity during development. In adult tecta, almost all microglial cells were of the mature ramified type and were distributed homogeneously in the different tectal layers, although in some layers they had particular morphological features. The distribution of microglia in the developing tectum and in adjacent regions provided insight into the routes of microglial cell invasion of the tectum during development. Apparently, a proportion of microglial cells reached the tectal parenchyma from the meninges and from the ventricular lumen, but the majority of them migrated along nerve fiber tracts from their entry point at the pial surface of the ventromedial caudal tectum. After they reached the stratum album centrale, microglial cells continued their migration toward more external layers, where they differentiated into ramified 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.

No abstract

Immunocytochemical techniques were used in conjunction with the QH1 antibody to study the morphological characteristics and distribution of microglia in the avascular retina of an avian species (the quail). The majority of microglial cells appeared in the outer and inner plexiform layers throughout the entire retina, whereas a few microglial cells in the nerve fiber layer were seen only in the central zone of the retina, near the optic nerve head. In the outer plexiform layer, microglial cells were star-shaped, with processes that ramified profusely in the horizontal plane. Fine process tips extended outward radially, insinuating themselves among the photoreceptors. A regular mosaic-like arrangement of microglial cells was evident in the outer plexiform layer, with no overlapping between adjacent cell territories. Microglial cells in the inner plexiform layer ramified through the entire width of this layer, showing radial and horizontal processes. Microglia in the inner plexiform layer also tended to be regularly distributed in a mosaic-like fashion, although there was slight overlapping between adjacent cell territories. Microglia density in this layer was approximately twice that in the outer plexiform layer. This pattern of microglial distribution was similar to that described in vascular retinae of several species of mammals, a finding that suggest that blood vessels are not responsible for the final locations of microglia in the adult retina, and that microglial precursors must migrate through long distances before they reach their precise destination.