FIGURE SUMMARY
Title

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

Authors
Iyer, H., Shen, K., Meireles, A.M., Talbot, W.S.
Source
Full text @ Sci Adv
Fig. 1

Fig. 1. Defective macrophages in rraga mutants fail to colonize the brain.
(A) Dorsal views of the midbrain showing mpeg:GFP expression at the indicated stages along with the corresponding quantification of developing microglia in the midbrain. WT, wild-type. (B) Representative images from time-lapse movies between 60 and ~78 hpf (dorsal views, anterior on top). Images show maximum intensity projections of z-slices at indicated times. (C to G) Quantitative analysis of time-lapse movies. In (C), each line shows macrophage cell counts from a single embryo over time. In (D), (E), and (G), each point represents counts from a single animal. (H to K) Lateral views of peripheral macrophages and quantification in rraga mutants and their siblings. (H, H′, and I) Macrophages in the head and (J, J′, and K) macrophages in the trunk + tail. Graphs in (A), (D) to (G), (I), and (K) show mean + SD; significance was determined using parametric unpaired t test. (L) EdU labeling to determine the percentage of proliferating macrophages in rraga mutants and wild-type siblings. Arrows note colocalization of EdU label with macrophages. Each point in the graph represents percentage of EdU+ macrophages from a single animal. Graph in (L) shows mean + SD; significance was determined using nonparametric Mann-Whitney U test. (M) High-magnification images showing the difference in the morphology of macrophages between rraga mutants and heterozygous animals at 3 dpf. (N) Quantification of amoeboid morphology of macrophages in rraga mutants. Graph in (N) shows mean + SD; significance was determined using nonparametric Mann-Whitney U test. The number of animals analyzed for each experiment is listed in table S1; all the panels are representative of at least two independent experiments. Scale bars, 50 μm.
Fig. 2

Fig. 2. Lysosomal genes are significantly up-regulated and endolysosomes are expanded in macrophages isolated from rraga mutants.
(A) Experimental schematic for RNA-seq. Macrophages were isolated using FACS from N = 90 larvae for each biological replicate RNA sample from rraga mutants or wild-type siblings. Four biological replicate samples of each genotype were processed for RNA-seq. (B) Volcano plot showing significantly differentially up-regulated and down-regulated genes in the macrophages of rraga mutants relative to wild-type siblings. (C) GO (Cellular Component) enrichment analysis showing an up-regulation of lysosome-associated terms in macrophages from rraga mutants. (D) Imaging and quantification of LAMP1 or LAMP2-mCherry transgene expression in rraga mutants and wild-type siblings at 4 dpf. Scale bars, 20 μm. (E) Imaging and quantification of mCherry-Rab5 or Rab7 transgene expression in rraga mutants and wild-type siblings at 4 dpf. Scale bars, 20 μm. (F) LysoSensor Green labeling and quantification of LysoSensor Green intensity at 4 dpf. Scale bars, 50 μm. Graphs in (D) to (F) show mean + SD; significance was determined using parametric unpaired t test. (G) DQ-BSA labeling at 4 dpf and quantification of DQ-BSA intensity inside macrophages. Scale bars, 50 μm. Graph shows mean + SD; significance was determined using nonparametric Mann-Whitney U test. The number of animals analyzed for each experiment is listed in table S1; all the panels (except RNA-seq) are representative of at least two independent experiments. A.U., arbitrary units.
Fig. 3

Fig. 3. Macrophages and microglia in rraga mutants show defective clearance of debris.
(A) Bar graph showing GO terms related to immune signaling significantly down-regulated in macrophages from rraga mutants. (B) Injection of E. coli Texas Red in rraga mutants and siblings at 4 dpf and corresponding quantification. Wild-type macrophages responding to E. coli become activated and display amoeboid morphology (asterisks). Scale bars, 50 μm. (C) GO terms related to migration/taxis significantly down-regulated in macrophages from rraga mutants. (D) Graphs showing fragments per kilobase of exon per million mapped reads (FPKM) values of csf1ra and csf1rb transcripts in macrophages from rraga mutants; each point represents FPKM value from a single RNA-seq biological replicate. FPKM, fragments per kilobase of exon per million mapped reads. (E and E′) Macrophage response to tail injury at 4 dpf. Arrows in the wild-type image show macrophages at the wound site (yellow dotted line). Scale bars, 20 μm. (F) TUNEL assay and quantification on rraga mutants and their wild-type siblings. Arrows indicate TUNEL+ microglia, and arrowheads indicate TUNEL signal outside microglia. Scale bars, 50 μm. (G to L) Time-lapse imaging and analysis of cellular characteristics of microglia at 5 dpf. (G) Images of rraga mutants and their siblings, with arrows showing phagocytic cup formation and arrowheads showing branching points. Scale bars, 20 μm. All graphs show mean + SD. Significance in (B) was determined using nonparametric Mann-Whitney U test; significance in (E′), (F), and (H) to (L) was determined using parametric unpaired t test. The number of animals analyzed for each experiment is listed in table S1; all the panels are representative of at least two independent experiments.
Fig. 4

Fig. 4. Microglia numbers are restored in rraga mutants with simultaneous mutation of tfeb, tfe3a, or tfe3b.
Images show dorsal views of the midbrain (anterior on top) of neutral red (NR)–stained larvae of indicated genotypes at 5 dpf, and graphs show corresponding quantification. (A and D) Progeny from rraga+/−; tfeb+/− intercross, (B and E) Progeny from rraga+/−; tfe3a+/− intercross. (C and F) Progeny from rraga+/−; tfe3b+/− intercross. (G to I) Progeny from rraga+/−; tfeb+/−; tfe3a+/−; tfe3b+/− quadruple heterozygous intercross. Images of a rraga mutant and tfeb and tfe3 genotypes that result in complete rescue of NR+ microglia in rraga mutants are shown in (H). In (G), the same genotypes, but in a background wild-type for rraga, are included for comparison. All graphs show mean + SD; significance was determined using parametric unpaired t test. The number of animals analyzed for each experiment is listed in the tables below each graph; (A) to (F) are representative of at least two independent experiments.
Fig. 5

Fig. 5. Simultaneous mutation in tfeb, tfe3a, or tfe3b rescues flcn and rraga mutant phenotypes.
(A) NR assay and quantification of microglia in larvae obtained from flcn+/−; tfeb+/−; tfe3b+/− intercross. (B and C) LysoTracker Red assay and quantification of the area of LysoTracker Red punctae in microglia of larvae from (B) rraga+/−; tfe3a+/− intercross and (C) flcn+/−; tfeb+/−; tfe3b+/− intercross. Images show LysoTracker Red signal in dorsal view of the midbrain. Scale bars, 50 μm. Graphs show mean + SD; significance was determined using parametric unpaired t test. (D) apoe mRNA expression at 4 dpf and rescue of microglia in the progeny of rraga+/−; tfe3a+/− intercross and flcn+/−; tfeb+/−; tfe3b+/− intercross. (E) mpeg:GFP expression to visualize microglia and macrophages in flcn; tfeb; tfe3b triple mutants. Arrows denote microglia and macrophages in which ramified morphology has been restored, asterisks denote cells with amoeboid morphology, and insets show magnified views of cell morphology. Scale bars, 50 μm. (F) Quantification of amoeboid morphology and rescue. Graph shows mean + SD; significance was determined using nonparametric Mann-Whitney U test. The number of animals analyzed for each experiment is listed in the tables below each graph; all the panels are representative of at least two independent experiments.

EXPRESSION / LABELING:
Genes:
Fish:
Anatomical Terms:
Stage: Day 4
PHENOTYPE:
Fish:
Observed In:
Stage Range: Day 4 to Day 5
Fig. 6

Fig. 6. Overexpression of tfe3b in the macrophage lineage disrupts microglia number and morphology as in rraga mutants.
Comparison of macrophages and microglia between animals overexpressing Tfe3b in the macrophage lineage, Tg(mpeg:tfe3b), and controls, Tg(mpeg:GFP), using (A) neutral red assay and quantification and (B) LysoTracker Red assay and quantification. Graphs show mean + SD; significance was determined using parametric unpaired t test. (C) apoe in situ hybridization and (D and E) live imaging with the mpeg:GFP transgene in (D) the brain and (E) the head, yolk, and tail regions of Tg(mpeg:tfe3b) and Tg(mpeg:GFP) larvae. Insets show magnified views of cell morphology. (F) Quantification of amoeboid morphology of microglia and macrophages. Graph shows mean + SD; significance was determined using nonparametric Mann-Whitney U test. Scale bars, 50 μm. The number of animals analyzed for each experiment is listed in table S1; all the panels are representative of at least two independent experiments. (G) Schematic summarizing the lysosomal regulatory circuit in microglia and macrophages.
Fig. 7

Fig. 7. Tfeb and Tfe3 activate lysosomal pathways only under conditions of stress.
(A) Microglia and macrophages visualized using the mpeg:GFP transgene in wild-type animals and tfeb; tfe3a; tfe3b triple mutants at 4 dpf. Macrophage response to (B) tail injury and (C) systemic ZymA injection in wild-type and triple mutant animals at 4 dpf. hpi, hours post-injury. Insets in (C) show magnified views of macrophage morphology. Graph in (B) shows mean + SD; significance was determined using parametric unpaired t test. The number of animals analyzed for each experiment is listed in table S1; all the panels are representative of at least two independent experiments. (D) Experimental schematic for RNA-seq. (E) Microscopy-based validation of ZymA Texas Red uptake by the trunk and tail macrophages in triple mutants and wild-type animals at 4 dpf before RNA-seq. Approximately 20 larvae of each genotype were used for RNA extraction per biological replicate; three biological replicates were used for RNA-seq. (F) Heatmap depicting all the lysosomal genes significantly up-regulated (log2 fold change > +1, Padj < 0.05) in rraga mutant whole larvae and the corresponding fold change of the gene in triple mutant larval RNA preparations. The log2 fold change is shown in each cell; fold change values with asterisks are not significant (Padj > 0.05). (G) GO term enrichment analysis of genes differentially up-regulated in wild-type ZymA-injected animals relative to uninjected wild-type controls. (H) GO term enrichment of genes significantly down-regulated in ZymA-injected triple mutants. FDR, false discovery rate.
Acknowledgments
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