In situ and transcriptomic identification of microglia in synapse-rich regions of the developing zebrafish brain

AbstractMicroglia 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 microglial subset in the synapse rich regions of the zebrafish (Danio rerio) brain. We found that ramified microglia increased in synaptic 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 synaptic-region associated microglia (SAMs) that were highly enriched in the hindbrain and expressed multiple candidate synapse modulating genes, including genes in the complement pathway. In contrast, neurogenic associated microglia (NAMs) were enriched in the optic tectum, had active cathepsin activity, and preferentially engulfed neuronal corpses. These data reveal that molecularly distinct phagocytic programs mediate synaptic remodeling and cell engulfment, and establish the zebrafish hindbrain as a model for investigating microglial-synapse interactions.

Download Full-text

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

10.1101/2021.05.08.443268 ◽

2021 ◽

Author(s):

Nicholas J Silva ◽

Leah C Dorman ◽

ilia vainchtein ◽

Nadine C Horneck ◽

Anna V Molofsky

Keyword(s):

Optic Tectum ◽

Apoptotic Cell ◽

Synaptic Proteins ◽

Mrna Sequencing ◽

Zebrafish Brain ◽

Synaptic Remodeling ◽

Cathepsin Activity ◽

Cell Engulfment ◽

Model Circuit

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.

Download Full-text

Faculty Opinions recommendation of Essential role of the apoptotic cell engulfment genes draper and ced-6 in programmed axon pruning during Drosophila metamorphosis.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1033559.387812 ◽

2006 ◽

Author(s):

Kai Zinn

Keyword(s):

Essential Role ◽

Apoptotic Cell ◽

Axon Pruning ◽

Cell Engulfment

Download Full-text

Two-color fluorescent in situ hybridization in the embryonic zebrafish brain using differential detection systems

BMC Developmental Biology ◽

10.1186/1471-213x-11-43 ◽

2011 ◽

Vol 11 (1) ◽

pp. 43 ◽

Cited By ~ 87

Author(s):

Gilbert Lauter ◽

Iris Söll ◽

Giselbert Hauptmann

Keyword(s):

In Situ Hybridization ◽

Fluorescent In Situ Hybridization ◽

Differential Detection ◽

Zebrafish Brain ◽

Detection Systems

Download Full-text

Critical Effects on Akt Signaling in Adult Zebrafish Brain Following Alterations in Light Exposure

Cells ◽

10.3390/cells10030637 ◽

2021 ◽

Vol 10 (3) ◽

pp. 637

Author(s):

Nicholas S. Moore ◽

Robert A. Mans ◽

Mackenzee K. McCauley ◽

Colton S. Allgood ◽

Keri A. Barksdale

Keyword(s):

Optic Tectum ◽

Visual Processing ◽

Environmental Enrichment ◽

Glycogen Synthase ◽

Light Exposure ◽

Hsp70 Expression ◽

Light Cycle ◽

Adult Zebrafish ◽

Sleep State ◽

Zebrafish Brain

Evidence from human and animal studies indicate that disrupted light cycles leads to alterations of the sleep state, poor cognition, and the risk of developing neuroinflammatory and generalized health disorders. Zebrafish exhibit a diurnal circadian rhythm and are an increasingly popular model in studies of neurophysiology and neuropathophysiology. Here, we investigate the effect of alterations in light cycle on the adult zebrafish brain: we measured the effect of altered, unpredictable light exposure in adult zebrafish telencephalon, homologous to mammalian hippocampus, and the optic tectum, a significant visual processing center with extensive telencephalon connections. The expression of heat shock protein-70 (HSP70), an important cell stress mediator, was significantly decreased in optic tectum of adult zebrafish brain following four days of altered light exposure. Further, pSer473-Akt (protein kinase B) was significantly reduced in telencephalon following light cycle alteration, and pSer9-GSK3β (glycogen synthase kinase-3β) was significantly reduced in both the telencephalon and optic tectum of light-altered fish. Animals exposed to five minutes of environmental enrichment showed significant increase in pSer473Akt, which was significantly attenuated by four days of altered light exposure. These data show for the first time that unpredictable light exposure alters HSP70 expression and dysregulates Akt-GSK3β signaling in the adult zebrafish brain.

Download Full-text

Molecular cloning and biochemical characterization of sialidases from zebrafish (Danio rerio)

Biochemical Journal ◽

10.1042/bj20070627 ◽

2007 ◽

Vol 408 (3) ◽

pp. 395-406 ◽

Cited By ~ 20

Author(s):

Marta Manzoni ◽

Paolo Colombi ◽

Nadia Papini ◽

Luana Rubaga ◽

Natascia Tiso ◽

...

Keyword(s):

Danio Rerio ◽

Biochemical Characterization ◽

Chromosome 21 ◽

Ph Optimum ◽

Reverse Transcription Pcr ◽

Genome Wide ◽

Genome Wide Search ◽

Putative Orthologues

Sialidases remove sialic acid residues from various sialo-derivatives. To gain further insights into the biological roles of sialidases in vertebrates, we exploited zebrafish (Danio rerio) as an animal model. A zebrafish transcriptome- and genome-wide search using the sequences of the human NEU polypeptides as templates revealed the presence of seven different genes related to human sialidases. neu1 and neu4 are the putative orthologues of the mammalian sialidases NEU1 and NEU4 respectively. Interestingly, the remaining genes are organized in clusters located on chromosome 21 and are all more closely related to mammalian sialidase NEU3. They were thus named neu3.1, neu3.2, neu3.3, neu3.4 and neu3.5. Using RT–PCR (reverse transcription–PCR) we detected transcripts for all genes, apart from neu3.4, and whole-mount in situ hybridization experiments show a localized expression pattern in gut and lens for neu3.1 and neu4 respectively. Transfection experiments in COS7 (monkey kidney) cells demonstrate that Neu3.1, Neu3.2, Neu3.3 and Neu4 zebrafish proteins are sialidase enzymes. Neu3.1, Neu3.3 and Neu4 are membrane-associated and show a very acidic pH optimum below 3.0, whereas Neu3.2 is a soluble sialidase with a pH optimum of 5.6. These results were further confirmed by subcellular localization studies carried out using immunofluorescence. Moreover, expression in COS7 cells of these novel zebrafish sialidases (with the exception of Neu3.2) induces a significant modification of the ganglioside pattern, consistent with the results obtained with membrane-associated mammalian sialidases. Overall, the redundancy of sialidases together with their expression profile and their activity exerted on gangliosides of living cells indicate the biological relevance of this class of enzymes in zebrafish.

Download Full-text

Survivin in relation to Bcl-2, Bax and in situ apoptotic cell death in anaplastic thyroid carcinoma

Archives of Biological Sciences ◽

10.2298/abs1104955s ◽

2011 ◽

Vol 63 (4) ◽

pp. 955-963

Author(s):

Sonja Selemetjev ◽

Dubravka Cvejic ◽

Svetlana Savin ◽

I. Paunovic ◽

S. Tatic

Keyword(s):

Cell Death ◽

Thyroid Carcinoma ◽

Apoptotic Cell ◽

Anaplastic Thyroid Carcinoma ◽

Apoptotic Cell Death ◽

Staining Score ◽

Human Malignancy ◽

Apoptotic Pathways ◽

Individual Scores

Anaplastic thyroid carcinoma (ATC) is a rare but highly aggressive human malignancy. It is known that disturbances in apoptotic pathways have a great impact on tumor progression and aggressiveness. In this study the apoptosisrelated molecules Bcl-2 (antiapoptotic), Bax (proapoptotic) and survivin (an inhibitor of apoptosis) were analyzed immunohistochemically in thirty archival cases of ATC. In situ apoptotic cell death was analyzed by the TUNEL method. Mean Bcl-2 staining score (calculated from individual scores from 0-3) was low compared to those for Bax and survivin (p<0.05). High expression of survivin was associated with high Bax expression, and was significantly segregated from high Bcl-2 expressing cases (p<0.05). Despite high Bax expression, apoptotic cell death was low in the investigated carcinomas. In addition, the mean apoptotic index in high survivin expressing carcinomas was significantly lower than in low survivin expressing carcinomas (p<0.05). It could be concluded that down-regulation of Bcl-2 is counterbalanced by up-regulation of survivin, which may overcome the effects of high Bax expression, and, at least partly, explain the low apoptosis rate and high biological aggressiveness of ATC.

Download Full-text

Impairment of Apoptotic Cell Engulfment by Pyocyanin, a Toxic Metabolite ofPseudomonas aeruginosa

American Journal of Respiratory and Critical Care Medicine ◽

10.1164/rccm.200612-1804oc ◽

2008 ◽

Vol 177 (1) ◽

pp. 35-43 ◽

Cited By ~ 73

Author(s):

Stephen M. Bianchi ◽

Lynne R. Prince ◽

Kathleen McPhillips ◽

Lucy Allen ◽

Helen M. Marriott ◽

...

Keyword(s):

Apoptotic Cell ◽

Toxic Metabolite ◽

Cell Engulfment

Download Full-text

Two Alternative Mechanisms That Regulate the Presentation of Apoptotic Cell Engulfment Signal in Caenorhabditis elegans

Molecular Biology of the Cell ◽

10.1091/mbc.e07-02-0138 ◽

2007 ◽

Vol 18 (8) ◽

pp. 3180-3192 ◽

Cited By ~ 73

Author(s):

Victor Venegas ◽

Zheng Zhou

Keyword(s):

Caenorhabditis Elegans ◽

Abc Transporter ◽

Germ Cells ◽

Mammalian Cells ◽

Developmental Stages ◽

Apoptotic Cell ◽

Somatic Cells ◽

Apoptotic Cells ◽

Phospholipid Scramblase ◽

Cell Engulfment

Phosphatidylserine exposed on the surface of apoptotic mammalian cells is considered an “eat-me” signal that attracts phagocytes. The generality of using phosphatidylserine as a clearance signal for apoptotic cells in animals and the regulation of this event remain uncertain. Using ectopically expressed mouse MFG-E8, a secreted phosphatidylserine-binding protein, we detected specific exposure of phosphatidylserine on the surface of apoptotic cells in Caenorhabditis elegans. Masking the surface phosphatidylserine inhibits apoptotic cell engulfment. CED-7, an ATP-binding cassette (ABC) transporter, is necessary for the efficient exposure of phosphatidylserine on apoptotic somatic cells, and for the recognition of these cells by phagocytic receptor CED-1. Alternatively, phosphatidylserine exposure on apoptotic germ cells is not CED-7 dependent, but instead requires phospholipid scramblase PLSC-1, a homologue of mammalian phospholipid scramblases. Moreover, deleting plsc-1 results in the accumulation of apoptotic germ cells but not apoptotic somatic cells. These observations suggest that phosphatidylserine might be recognized by CED-1 and act as a conserved eat-me signal from nematodes to mammals. Furthermore, the two different biochemical activities used in somatic cells (ABC transporter) and germ cells (phospholipid scramblase) suggest an increased complexity in the regulation of phosphatidylserine presentation in response to apoptotic signals in different tissues and during different developmental stages.

Download Full-text