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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.

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.

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 Muller 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 E 14. 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. © 1995 Wiley-Liss, Inc.