PUBLICATION

A lysosomal regulatory circuit essential for the development and function of microglia

Authors: Iyer, H., Shen, K., Meireles, A.M., Talbot, W.S.
ID: ZDB-PUB-220901-6
Date: 2022
Source: Science advances 8: eabp8321 (Journal)
Registered Authors: Talbot, William S.
Keywords: none
Datasets: GEO:GSE197350, GEO:GSE197349, GEO:GSE197348
MeSH Terms: none
PubMed: 36044568 Full text @ Sci Adv

Abstract

As the primary phagocytic cells of the central nervous system, microglia exquisitely regulate their lysosomal activity to facilitate brain development and homeostasis. However, mechanisms that coordinate lysosomal activity with microglia development, chemotaxis, and function remain unclear. Here, we show that embryonic macrophages require the lysosomal guanosine triphosphatase (GTPase) RagA and the GTPase-activating protein Folliculin to colonize the brain in zebrafish. We demonstrate that embryonic macrophages in rraga mutants show increased expression of lysosomal genes but display significant down-regulation of immune- and chemotaxis-related genes. Furthermore, we find that RagA and Folliculin repress the key lysosomal transcription factor Tfeb and its homologs Tfe3a and Tfe3b in the macrophage lineage. Using RNA sequencing, we establish that Tfeb and Tfe3 are required for activation of lysosomal target genes under conditions of stress but not for basal expression of lysosomal pathways. Collectively, our data define a lysosomal regulatory circuit essential for macrophage development and function in vivo.

Genes / Markers

Figures

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Expression

Gene	Fish	Conditions	Stage	Anatomy	Assay	Figure
apoeb	flcn^st127/st127	standard conditions	Day 4	microglial cell	ISH	Fig. 5
apoeb	gl22Tg/gl22Tg; zf3682Tg/zf3682Tg	standard conditions	Day 4	midbrain microglial cell	ISH	Fig. 6
apoeb	rraga^st77/st77	standard conditions	Day 4	midbrain microglial cell	ISH	Fig. 5
apoeb	rraga^st77/st77; tfe3a^st124/st124	standard conditions	Day 4	microglial cell	ISH	Fig. 5
apoeb	rraga^st77/+; tfe3a^st124/st124	standard conditions	Day 4	microglial cell	ISH	Fig. 5
apoeb	tfe3b^st125/st125; tfeb^st120/st120; flcn^st127/st127	standard conditions	Day 4	microglial cell	ISH	Fig. 5
apoeb	tfe3b^st125/st125; tfeb^st120/st120; flcn^st127/+	standard conditions	Day 4	microglial cell	ISH	Fig. 5
csf1ra	rraga^st77/st77; gl22Tg/gl22Tg	standard conditions	Day 4	macrophage	RNA-Seq	Fig. 3
csf1rb	rraga^st77/st77; gl22Tg/gl22Tg	standard conditions	Day 4	macrophage	RNA-Seq	Fig. 3
EGFP	flcn^st127/st127; gl22Tg/gl22Tg	standard conditions	Day 4	macrophage	IFL	Fig. 5

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Phenotype

Fish	Conditions	Stage	Phenotype	Figure
flcn^st127/st127	standard conditions	Day 4	microglial cell apoeb expression decreased amount, abnormal	Fig. 5
flcn^st127/st127	standard conditions	Day 5	microglial cell lysosomal lumen acidification increased occurrence, abnormal	Fig. 5
flcn^st127/st127; gl22Tg/gl22Tg	standard conditions	Day 4	macrophage EGFP expression spatial pattern, abnormal	Fig. 5
flcn^st127/st127; gl22Tg/gl22Tg	standard conditions	Day 4	microglial cell EGFP expression spatial pattern, abnormal	Fig. 5
gl22Tg/gl22Tg	chemical treatment by environment: (1->3)-beta-D-glucan	Day 4	macrophage inflammatory response increased occurrence, abnormal	Fig. 7
gl22Tg/gl22Tg	chemical treatment by environment: (1->3)-beta-D-glucan	Day 4	macrophage lysosomal enzyme production involved in inflammatory response decreased rate, abnormal	Fig. 7
gl22Tg/gl22Tg	chemical treatment by environment: (1->3)-beta-D-glucan	Day 4	macrophage response to toxic substance increased occurrence, abnormal	Fig. 7
gl22Tg/gl22Tg	chemical treatment by environment: (1->3)-beta-D-glucan	Day 4	macrophage response to xenobiotic stimulus increased occurrence, abnormal	Fig. 7
gl22Tg/gl22Tg	bacterial treatment by exposure to environment: Escherichia coli	Day 4	macrophage circular, abnormal	Fig. 3
gl22Tg/gl22Tg; zf3682Tg/zf3682Tg	standard conditions	Day 4	caudal fin macrophage circular, abnormal	Fig. 6

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Mutations / Transgenics

Allele	Construct	Type	Affected Genomic Region
gl22Tg	Tg(mpeg1:EGFP)	Transgenic Insertion
st77		Point Mutation	rraga
st120		Small Deletion	tfeb
st124		Small Deletion	tfe3a
st125		Small Deletion	tfe3b
st126		Small Deletion	tfe3b
st127		Complex	flcn
zf3682Tg	Tg(mpeg1.1:tfe3b,myl7:mCherry)	Transgenic Insertion
zf3683Tg	Tg(mpeg1.1:rab5c-mCherry,myl7:EGFP)	Transgenic Insertion
zf3684Tg	Tg(mpeg1.1:rab7a-mCherry,myl7:EGFP)	Transgenic Insertion

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Human Disease / Model

Sequence Targeting Reagents

Target	Reagent	Reagent Type
adgrb1b	MO1-adgrb1b	MRPHLNO
adgrb1b	MO2-adgrb1b	MRPHLNO
casp3a	MO1-casp3a	MRPHLNO
flcn	CRISPR2-flcn	CRISPR
flcn	CRISPR1-flcn	CRISPR
havcr1	MO1-havcr1	MRPHLNO
havcr1	MO2-havcr1	MRPHLNO
tfe3a	CRISPR1-tfe3a	CRISPR
tfe3b	CRISPR1-tfe3b	CRISPR
tfe3b	CRISPR2-tfe3b	CRISPR

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Fish

Fish
flcn^st127/st127
flcn^st127/st127; gl22Tg/gl22Tg
gl22Tg/gl22Tg
gl22Tg/gl22Tg; zf3682Tg/zf3682Tg
rraga^st77/st77
rraga^st77/st77; gl22Tg/gl22Tg
rraga^st77/st77; tfe3a^st124/st124
rraga^st77/st77; tfe3a^st124/st124; tfe3b^st125/st125; tfeb^st120/st120
rraga^st77/st77; tfe3a^st124/+
rraga^st77/st77; tfe3a^st124/+; tfe3b^st125/+; tfeb^st120/+

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Antibodies

Orthology

Engineered Foreign Genes

Marker	Marker Type	Name
EGFP	EFG	EGFP
mCherry	EFG	mCherry

Mapping

Iyer et al., 2022 ZDB-PUB-220901-6 PMID:36044568

A lysosomal regulatory circuit essential for the development and function of microglia

Iyer et al., 2022

ZDB-PUB-220901-6
PMID:36044568