In situand transcriptomic identification of synapse-associated microglia in the developing zebrafish brain

Abstract

Microglia are brain resident macrophages that play vital roles in central nervous system (CNS) development, homeostasis, and pathology. Microglia both remodel synapses and engulf apoptotic cell corpses during development, but whether unique molecular programs regulate these distinct phagocytic functions is unknown. Here we identify a molecularly distinct synapse-associated microglial subset in the zebrafish (Danio rerio). We found that ramified microglia populated synapse-rich regions of the midbrain and hindbrain between 7 and 28 days post fertilization. In contrast, microglia in the optic tectum were ameboid and clustered around neurogenic zones. Using single-cell mRNA sequencing combined with metadata from regional bulk sequencing, we identified synapse-associated microglia (SAMs) that were highly enriched in the hindbrain, expressed known synapse modulating genes as well as novel candidates, and engulfed synaptic proteins. In contrast, neurogenic-associated microglia (NAMs) were enriched in optic tectum, had active cathepsin activity, and preferentially engulfed neuronal corpses. These data yielded a functionally annotated atlas of zebrafish microglia (https://www.annamolofskylab.org/microglia-sequencing). Furthermore, they reveal that molecularly distinct phagocytic programs mediate synaptic remodeling and cell engulfment, and establish zebrafish hindbrain as a model circuit for investigating microglial-synapse interactions.

Insights Into Central Nervous System Glial Cell Formation and Function From Zebrafish

“…These migrate to the hematopoietic tissue [the rostral blood island (RBI)], and then from peripheral tissues to the optic tectum during early embryonic development ( Herbomel, 1999 ). A second wave, which derives from hematopoietic stem cells from the ventral wall of the dorsal aorta replaces the original population later in development ( Ferrero et al, 2018 ; Wu et al, 2020 ; Silva et al, 2021 ). A number of conserved transcription factors have been shown to be required for microglia development in zebrafish and mammalian models including Pu.1 and Irf8 ( Rhodes et al, 2005 ; Li et al, 2011 ; Shiau et al, 2015 ) ( Supplementary Table 1 ).…”

“…That being said, there is evidence to suggest that microglia in the zebrafish may play important roles in synaptogenesis. For example, a recent study from Silva et al (2021) describe a novel subpopulation of synapse-associated microglia present in the midbrain and hindbrain. Synapse-associated microglia express the complement gene C1qc, a known protein which also regulates synaptic engulfment by microglia in rodents ( Stevens et al, 2007 ; Hong et al, 2016 ), which could suggest that it may mediate similar functions in the zebrafish.…”

A Model of Discovery: The Role of Imaging Established and Emerging Non-mammalian Models in Neuroscience

“…Microglia in zebrafish are a rapidly expanding topic of interest–more papers have been published between 2019 and 2021 on microglia in zebrafish that in the preceding 12 years combined. Zebrafish microglia are generated in two waves, with a primitive wave derived from the primitive macrophages in the yolk sac, and a second wave that replaces the original population and originates from the hematopoietic stem cells of the dorsal aorta ( Xu et al, 2015 ; Ferrero et al, 2018 ; Wu et al, 2020 ; Silva et al, 2021 ). Zebrafish microglia share a core transcriptional signature with mammalian microglia and are morphologically and functionally similar to microglia of other species ( Oosterhof et al, 2017 ; Geirsdottir et al, 2019 ).…”