José L. Marín-Teva

Phagocytosis in the developing CNS: more than clearing the corpses.

Cell corpses generated during CNS development are eliminated through phagocytosis performed by a variety of cells, including mesenchyme-derived macrophages and microglia, or glial cells originating in the neurogenic ectoderm. Mounting evidence indicates that in different species, phagocytes not only clear cell corpses but also engulf still-living neural cells or axons, and thereby promote cell death or axon pruning. Knowledge of the mechanisms of corpse recognition by engulfing cells provides molecular signals to this new role for phagocytes. These observations support a conserved and instructive role for phagocytosis in the execution of regressive events during neurogenesis.

Embryonic and postnatal development of microglial cells in the mouse retina

Macrophage/microglial cells in the mouse retina during embryonic and postnatal development were studied by immunocytochemistry with Iba1, F4/80, anti‐CD45, and anti‐CD68 antibodies and by tomato lectin histochemistry. These cells were already present in the retina of embryos aged 11.5 days (E11.5) in association with cell death. At E12.5 some macrophage/microglial cells also appeared in peripheral regions of the retina with no apparent relationship with cell death. Immediately before birth microglial cells were present in the neuroblastic, inner plexiform (IPL), and ganglion cell (GCL) layers, and their distribution suggested that they entered the retina from the ciliary margin and the vitreous. The density of retinal microglial cells strongly decreased at birth, increased during the first postnatal week as a consequence of the entry of microglial precursors into the retina from the vitreous, and subsequently decreased owing to the cessation of microglial entry and the increase in retina size. The mature topographical distribution pattern of microglia emerged during postnatal development of the retina, apparently by radial migration of microglial cells from the vitreal surface in a vitreal‐to‐scleral direction. Whereas microglial cells were only seen in the GCL and IPL at birth, they progressively appeared in more scleral layers at increasing postnatal ages. Thus, microglial cells were present within all layers of the retina except the outer nuclear layer at the beginning of the second postnatal week. Once microglial cells reached their definitive location, they progressively ramified. J. Comp. Neurol. 506:224–239, 2008.

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. J. Comp. Neurol. 412:255–275, 1999.

Microglia and neuronal cell death

Microglia, the brains innate immune cell type, are cells of mesodermal origin that populate the central nervous system (CNS) during development. Undifferentiated microglia, also called ameboid microglia, have the ability to proliferate, phagocytose apoptotic cells and migrate long distances toward their final destinations throughout all CNS regions, where they acquire a mature ramified morphological phenotype. Recent studies indicate that ameboid microglial cells not only have a scavenger role during development but can also promote the death of some neuronal populations. In the mature CNS, adult microglia have highly motile processes to scan their territorial domains, and they display a panoply of effects on neurons that range from sustaining their survival and differentiation contributing to their elimination. Hence, the fine tuning of these effects results in protection of the nervous tissue, whereas perturbations in the microglial response, such as the exacerbation of microglial activation or lack of microglial response, generate adverse situations for the organization and function of the CNS. This review discusses some aspects of the relationship between microglial cells and neuronal death/survival both during normal development and during the response to injury in adulthood.

Microglial response to light-induced photoreceptor degeneration in the mouse retina

The microglial response elicited by degeneration of retinal photoreceptor cells was characterized in BALB/c mice exposed to bright light for 7 hours and then kept in complete darkness for survival times ranging from 0 hours to 10 days. Photodegeneration resulted in extensive cell death in the retina, mainly in the outer nuclear layer (ONL), where the photoreceptor nuclei are located. Specific immunolabeling of microglial cells with anti‐CD11b, anti‐CD45, anti‐F4/80, anti‐SRA, and anti‐CD68 antibodies revealed that microglial cells were activated in light‐exposed retinas. They migrated to the ONL, changed their morphology, becoming rounded cells with short and thick processes, and, finally, showed immunophenotypic changes. Specifically, retinal microglia began to strongly express antigens recognized by anti‐CD11b, anti‐CD45, and anti‐F4/80, coincident with cell degeneration. In contrast, upregulation of the antigen recognized by anti‐SRA was not detected by immunocytochemistry until 6 hours after light exposure. Differences were also observed at 10 days after light exposure: CD11b, CD45, and F4/80 continued to be strongly expressed in retinal microglia, whereas the expression of CD68 and SRA had decreased to near‐normal values. Therefore, microglia did not return to their original state after photodegeneration and continued to show a degree of activation. The accumulation of activated microglial cells in affected regions simultaneously with photoreceptor degeneration suggests that they play some role in photodegeneration. J. Comp. Neurol. 518:477–492, 2010.

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.

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. GLIA 22:31–52, 1998.

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.

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-β-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.

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. J. Comp. Neurol. 389:390–401, 1997.