Antonio Almendros

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.

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.

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.

Alterations in the digestive gland and shell of the snail Helix aspersa Müller (gastropoda, pulmonata) after prolonged starvation

We investigated the effects of prolonged starvation on body weight and shell thickness, on the numbers of different types of cells, and on the Mg/Ca ratio in the digestive gland and shell in the common garden snail Helix mprsa Miiller. A 60% reduction in body weight, which occurred after periods of starvation longer than 3 months, was critical for life. Starvation had a marked effect on the relative numbers of cells in the digestive gland, and significantly decreased the number of digestive cells. The changes in Mg/Ca ratio in the digestive gland and shell were closely correlated. Changes in ion balance may have resulted from the need to compensate for the starvation-induced decline in extracellular pH. The shell acted as a reservoir of ions that could be mobilized under extreme conditions. COMP BIOCHEM PHYSIOL 115A;l:ll-17, 1996.

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. GLIA 27:226–238, 1999.

Localization and distribution of alkaline phosphatase activity in the hepatopancreas of the snail

Histocytochemical methods were used to investigate alkaline phosphatase activity in the digestive gland (hepatopancreas) of the common garden snail Helix aspersa. Histochemical findings and light microscopic observations showed that enzymatic activity was confined mainly to the basal connective tissue that enveloped the adenomeres. Transmission electron microscopy showed that enzymatic activity was localized in the plasma membrane, and showed an intercellular distribution along the lateral surfaces and the basal portions of the cells in different adenomeres. Alkaline phosphatase activity was also found in the plasma membrane of fibrocytes of the basal connective tissue enveloping the adenomeres. Enzymatic activity was seen around the fat droplets of glandular cells. The possible involvement of alkaline phosphatase in processes or remodelling of the basal connective tissue that envelopes the gland is discussed.