Onset of microglial entry into developing quail retina coincides with increased expression of active caspase-3 and is mediated by extracellular ATP and UDP.

María Martín-Estebané,María-Carmen Carrasco,Julio Navascués,José L Marín-Teva,Ana Sierra-Martín,Miguel A Cuadros,Sandra M Martín-Guerrero,Fernando De Castro

doi:10.1371/journal.pone.0182450

María Martín-Estebané, María-Carmen Carrasco + Show 6 more

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https://doi.org/10.1371/journal.pone.0182450

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Abstract

Microglial cell precursors located in the area of the base of the pecten and the optic nerve head (BP/ONH) start to enter the retina of quail embryos at the 7th day of incubation (E7), subsequently colonizing the entire retina by central-to-peripheral tangential migration, as previously shown by our group. The present study demonstrates a precise chronological coincidence of the onset of microglial cell entry into the retina with a striking increase in death of retinal cells, as revealed by their active caspase-3 expression and TUNEL staining, in regions dorsal to the BP/ONH area, suggesting that dying retinal cells would contribute to the microglial cell inflow into the retina. However, the molecular mechanisms involved in this inflow are currently unclear. Extracellular nucleotides, such as ATP and UDP, have previously been shown to favor migration of microglia towards brain injuries because they are released by apoptotic cells and stimulate both chemotaxis and chemokinesis in microglial cells via signaling through purinergic receptors. Hence, we tested here the hypothesis that ATP and UDP play a role in the entry and migration of microglial precursors into the developing retina. For this purpose, we used an experimental model system based on organotypic cultures of E6.5 quail embryo retina explants, which mimics the entry and migration of microglial precursors in the in situ developing retina. Inhibition of purinergic signaling by treating retina explants with either apyrase, a nucleotide-hydrolyzing enzyme, or suramin, a broad spectrum antagonist of purinergic receptors, significantly prevents the entry of microglial cells into the retina. In addition, treatment of retina explants with either exogenous ATP or UDP results in significantly increased numbers of microglial cells entering the retina. In light of these findings, we conclude that purinergic signaling by extracellular ATP and UDP is necessary for the entry and migration of microglial cells into the embryonic retina by inducing chemokinesis in these cells.

Highlights

Microglia are resident macrophages of the central nervous system (CNS) that derive from myeloid hematopoietic progenitors [1,2,3]
Immunofluorescence analysis revealed that active caspase-3 expression was very low in the quail embryo retina at E6 (Fig 1A and 1B), when microglial cells had not begun to enter the retina (Fig 1C), but was markedly increased at E7 (Fig 1D and 1E), when the first microglial cells were entering the retina from the BP/ONH area (Fig 1F)
Our group previously showed that microglial cells in the quail retina derive from precursors of amoeboid phenotype that start to enter the embryonic retina from the BP/ONH area at E7 and migrate tangentially in a central-to-peripheral direction by a similar cellular mechanism to that of cultured fibroblasts in vitro [15,16,17]

Summary

Introduction

Microglia are resident macrophages of the central nervous system (CNS) that derive from myeloid hematopoietic progenitors [1,2,3]. We used our in vitro model system based on organotypic cultures of quail embryo retina explants to show that purinergic signaling by extracellular ATP and UDP, presumably released from dying retinal cells, is involved in the entry and chemokinetic migration of microglial cells into the retina, similar to observations in the developing zebrafish brain. This mechanism for microglial colonization of the CNS appears to be a conserved process across vertebrate species

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