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A transgenic inducible GFP extracellular-vesicle reporter (TIGER) mouse illuminates neonatal cortical astrocytes as a source of immunomodulatory extracellular vesicles

Neckles, Victoria N., Morton, Mary C., Holmberg, Jennie C., Sokolov, Aidan M., Nottoli, Timothy, Liu, Don, Feliciano, David M.·2019·Scientific Reports
DOI 10.1038/s41598-019-39679-0PMC PMC6395689
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Abstract

Extracellular vesicles (EVs) are cellular derived particles found throughout the body in nearly all tissues and bodily fluids. EVs contain biological molecules including small RNAs and protein. EVs are proposed to be transferred between cells, notably, cells of the immune system. Tools that allow for in vivo EV labeling while retaining the ability to resolve cellular sources and timing of release are required for a full understanding of EV functions. Fluorescent EV fusion proteins are useful for the study of EV biogenesis, release, and identification of EV cellular recipients. Among the most plentiful and frequently identified EV proteins is CD9, a tetraspanin protein. A transgenic mouse containing a CRE-recombinase inducible CAG promoter driven CD9 protein fused to Turbo-GFP derived from the copepod Pontellina plumata was generated as an EV reporter. The transgenic inducible GFP EV reporter (TIGER) mouse was electroporated with CAG-CRE plasmids or crossed with tamoxifen inducible CAG-CRE-ER T2 or nestin-CRE-ER T2 mice. CD9-GFP labeled cells included glutamine synthetase and glial fibrillary acidic protein positive astrocytes. Cortical astrocytes released ~136 nm EVs that contained CD9. Intraventricular injected EVs were taken up by CD11b/IBA1 positive microglia surrounding the lateral ventricles. Neonatal electroporation and shRNA mediated knockdown of Rab27a in dorsal subventricular zone NSCs and astrocytes increased the number of CD11b/IBA1 positive rounded microglia. Neonatal astrocyte EVs had a unique small RNA signature comprised of morphogenic miRNAs that induce microglia cytokine release. The results from this study demonstrate that inducible CD9-GFP mice will provide the EV community with a tool that allows for EV labeling in a cell-type specific manner while simultaneously allowing in vivo experimentation and provides evidence that EVs are required immunomodulators of the developing nervous system.

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Crossref · 10.1038/s41598-019-39679-0

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Figures

Figure 1 (A) Schematic of mouse genotype. (B) Image of dissected tissue from CAG-CRE-ERT2 × TIGER mouse cross following 100 μg/g tamoxifen injection at P2 and sacrificed at P4. H, heart, B, brain, LV, liver, S, stomach, K, Kidney. Scale bar, 1 cm. (C) Depth coded volumetric 2 photon microscopy image of CD9-GFP following 100 μg/g tamoxifen injection starting at P2 until sacrifice at P8 in a spleen following CLARITY. Color indicates depth of imaging. Scale bar, 50 μm. (D) Cultured fibroblast treated with tamoxifen from TIGER × CAG-CRE-ERT2 mice. Scale bar, 12.5 μm (E–H) Sections of TIGER × CAG-CRE-ERT2 mouse tissue following 100 μg/g tamoxifen at P2 and sacrificed at P4. (E) Coronal section of TIGER × CAG-CRE-ERT2 mouse near ventricle showing CD9-GFP positive cells. Scale bar, 25 μm. (F) Section of stomach of a TIGER × CAG-CRE-ERT2 mouse. Scale bar, 25 μm. (G) Section of a heart of a CD9-GFP × CAG-CRE-ERT2 mouse. (H) Section of kidney of a TIGER × CAG-CRE-ERT2 mouse. Scale bar, 25 μm.
Figure 1 (A) Schematic of mouse genotype. (B) Image of dissected tissue from CAG-CRE-ERT2 × TIGER mouse cross following 100 μg/g tamoxifen injection at P2 and sacrificed at P4. H, heart, B, brain, LV, liver, S, stomach, K, Kidney. Scale bar, 1 cm. (C) Depth coded volumetric 2 photon microscopy image of CD9-GFP following 100 μg/g tamoxifen injection starting at P2 until sacrifice at P8 in a spleen following CLARITY. Color indicates depth of imaging. Scale bar, 50 μm. (D) Cultured fibroblast treated with tamoxifen from TIGER × CAG-CRE-ERT2 mice. Scale bar, 12.5 μm (E–H) Sections of TIGER × CAG-CRE-ERT2 mouse tissue following 100 μg/g tamoxifen at P2 and sacrificed at P4. (E) Coronal section of TIGER × CAG-CRE-ERT2 mouse near ventricle showing CD9-GFP positive cells. Scale bar, 25 μm. (F) Section of stomach of a TIGER × CAG-CRE-ERT2 mouse. Scale bar, 25 μm. (G) Section of a heart of a CD9-GFP × CAG-CRE-ERT2 mouse. (H) Section of kidney of a TIGER × CAG-CRE-ERT2 mouse. Scale bar, 25 μm.CC BY · PubMed Central
Figure 2 (A) Depth coded volumetric rendering of cerebral cortex of a TIGER × Nestin-CRE-ERT2 mouse. Scale bar, 50 μm. (B–D) CD9-GFP (green) × CAG-CRE-ERT2 brain sectioned and stained for GFAP (red) and counter-stained with TOPRO (blue) to label nuclei merged (B), CD9-GFP alone (C), and CD9-GFP/GFAP (D). Arrows point to cells with co-localization. Scale bar, 100 μm. (E,F) 20X image of CD9-GFP (green) CAG-CRE-ERT2 brain sections stained for GFAP (red) and counter-stained with (E) or without (F) TOPRO (blue). Arrows point to colocalization. Scale bar, 50 μm. (G,H) Image showing CD9-GFP (green) from TIGER × CAG CRE ERT2 brain sections stained for GS (red) and TOPRO (blue) (G) or CD9-GFP only (H). Arrows points to colocalization. Scale bar, 50 μm. (I) 63X image of cortex demonstrating CD9-GFP in astrocytes. Scale bar, 25 μm. (J) Astrocyte culture from TIGER × Tomato mice electroporated with CRE and counter-stained with TOPRO (blue). Scale bar, 50 μm. (K) Cultured astrocytes from TIGER mice and counter-stained with TOPRO (blue). Scale bar, 12.5 μm. (L) CD9-GFP (green) and tomato (red) electroporated astrocyte cultures to demonstrate morphology. Scale bar, 25 μm.
Figure 2 (A) Depth coded volumetric rendering of cerebral cortex of a TIGER × Nestin-CRE-ERT2 mouse. Scale bar, 50 μm. (B–D) CD9-GFP (green) × CAG-CRE-ERT2 brain sectioned and stained for GFAP (red) and counter-stained with TOPRO (blue) to label nuclei merged (B), CD9-GFP alone (C), and CD9-GFP/GFAP (D). Arrows point to cells with co-localization. Scale bar, 100 μm. (E,F) 20X image of CD9-GFP (green) CAG-CRE-ERT2 brain sections stained for GFAP (red) and counter-stained with (E) or without (F) TOPRO (blue). Arrows point to colocalization. Scale bar, 50 μm. (G,H) Image showing CD9-GFP (green) from TIGER × CAG CRE ERT2 brain sections stained for GS (red) and TOPRO (blue) (G) or CD9-GFP only (H). Arrows points to colocalization. Scale bar, 50 μm. (I) 63X image of cortex demonstrating CD9-GFP in astrocytes. Scale bar, 25 μm. (J) Astrocyte culture from TIGER × Tomato mice electroporated with CRE and counter-stained with TOPRO (blue). Scale bar, 50 μm. (K) Cultured astrocytes from TIGER mice and counter-stained with TOPRO (blue). Scale bar, 12.5 μm. (L) CD9-GFP (green) and tomato (red) electroporated astrocyte cultures to demonstrate morphology. Scale bar, 25 μm.CC BY · PubMed Central
Figure 3 (A) Quantification of CD9 protein relative to AKT in cortical lysates at the indicated ages according to western blots of dorsal cortical lysates for CD9 and AKT as a loading control. The ~37 kDa (black bar) and 20–25 kDa (grey bar) CD9 bands were quantified. N = 3 independent biological replicates. (B) RNA sequencing reads for CD9 based on cell type listed. Astro. = Astrocyte, Endoth. = Endothelial cell, Micro = microglia, Oligo Stem = Oligodendrocyte stem cell, Oligo New = immature oligodendrocyte, Oligo Myelin = Mature myelinating oligodendrocyte. (C) Quantification of astrocyte CD9 mRNA associated with active ribosomes from different regions and ages. Visual = visual cortex, Motor = Motor cortex, Somat. = Somatosensory cortex, Hypoth. = Hypothalamus, Cereb. = Cerebellum. (D) Western blots of astrocyte lysate and EVs for CD9 and CD63. (E) Nanosight particle tracking analysis frequency histogram of astrocyte conditioned media. Data are represented as mean ± SEM.
Figure 3 (A) Quantification of CD9 protein relative to AKT in cortical lysates at the indicated ages according to western blots of dorsal cortical lysates for CD9 and AKT as a loading control. The ~37 kDa (black bar) and 20–25 kDa (grey bar) CD9 bands were quantified. N = 3 independent biological replicates. (B) RNA sequencing reads for CD9 based on cell type listed. Astro. = Astrocyte, Endoth. = Endothelial cell, Micro = microglia, Oligo Stem = Oligodendrocyte stem cell, Oligo New = immature oligodendrocyte, Oligo Myelin = Mature myelinating oligodendrocyte. (C) Quantification of astrocyte CD9 mRNA associated with active ribosomes from different regions and ages. Visual = visual cortex, Motor = Motor cortex, Somat. = Somatosensory cortex, Hypoth. = Hypothalamus, Cereb. = Cerebellum. (D) Western blots of astrocyte lysate and EVs for CD9 and CD63. (E) Nanosight particle tracking analysis frequency histogram of astrocyte conditioned media. Data are represented as mean ± SEM.CC BY · PubMed Central
Figure 4 (A–D) Images of coronal brain sections showing DiI (red) labeled EVs taken up by IBA1 (blue) and CD11b (green) positive microglia. (E,F) Tomato and control plasmid (blue) dorsal SVZ electroporations stained for CD11b (green) and IBA1 (magenta) at P7, 10x (E), 20x (F). (G,H) 20x magnification of E showing CD11b (green) and IBA1 (red) (G) or CD11b (H). (I,J) Tomato and Rab27a shRNA plasmid (blue) dorsal SVZ electroporations stained for CD11b (green) and IBA1 (magenta) at P7, 10x (I), 20x (J). (K,L) 20x magnification of I showing CD11b (green) and IBA1 (red) (K) or CD11b (L). (M) Western blot of Rab27a following shRNA mediated knockdown. (N) Images of coronal section stained for Rab27a (green) following control or Rab27a shRNA and tomato (magenta) co-electroporation at P0 and sacrificed 48 hrs later. (O) Quantification of CD11b positive microglia at P2 and P7 following control or Rab27a shRNA electroporation. Data are represented as mean ± SEM. ****p < 0.0001 A, E, I scale bar, 100 μm. B–D, F–H, J–L, N scale bar, 50 μm.
Figure 4 (A–D) Images of coronal brain sections showing DiI (red) labeled EVs taken up by IBA1 (blue) and CD11b (green) positive microglia. (E,F) Tomato and control plasmid (blue) dorsal SVZ electroporations stained for CD11b (green) and IBA1 (magenta) at P7, 10x (E), 20x (F). (G,H) 20x magnification of E showing CD11b (green) and IBA1 (red) (G) or CD11b (H). (I,J) Tomato and Rab27a shRNA plasmid (blue) dorsal SVZ electroporations stained for CD11b (green) and IBA1 (magenta) at P7, 10x (I), 20x (J). (K,L) 20x magnification of I showing CD11b (green) and IBA1 (red) (K) or CD11b (L). (M) Western blot of Rab27a following shRNA mediated knockdown. (N) Images of coronal section stained for Rab27a (green) following control or Rab27a shRNA and tomato (magenta) co-electroporation at P0 and sacrificed 48 hrs later. (O) Quantification of CD11b positive microglia at P2 and P7 following control or Rab27a shRNA electroporation. Data are represented as mean ± SEM. ****p < 0.0001 A, E, I scale bar, 100 μm. B–D, F–H, J–L, N scale bar, 50 μm.CC BY · PubMed Central
Figure 5 (A) Top most abundant miRNA reads from astrocyte EVs. (B) Hierarchical clustering analysis of small RNAs from neonatal NSC and astrocyte EVs. (C) Statistically significant up and down-regulated miRNAs. (D) Differential expression profile of individual miRNAs. (E) Cytokine profiles from microglia condition media. Data are represented as mean ± SEM. *p < 0.05; **p < 0.01; ****p < 0.0001.
Figure 5 (A) Top most abundant miRNA reads from astrocyte EVs. (B) Hierarchical clustering analysis of small RNAs from neonatal NSC and astrocyte EVs. (C) Statistically significant up and down-regulated miRNAs. (D) Differential expression profile of individual miRNAs. (E) Cytokine profiles from microglia condition media. Data are represented as mean ± SEM. *p < 0.05; **p < 0.01; ****p < 0.0001.CC BY · PubMed Central

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Citations

Cites

Research works printed but not linked: 28

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Metadata

Authors
Neckles, V. N., Morton, M. C., Holmberg, J. C., Sokolov, A. M., Nottoli, T., Liu, D., & Feliciano, D. M.
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2019-02-28
Volume
9
Issue
1
ISSN
2045-2322
DOI
10.1038/s41598-019-39679-0
PMC
PMC6395689
Cited by
55
References
6
Language
en
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