Pdgfra marks a cellular lineage with distinct contributions to myofibroblasts in lung maturation and injury response
- University of California, San Diego, United States
- University of Wisconsin-Madison, United States
Figures
Figure 1 with 1 supplement
Generation and characterization of PdgfrartTA knock-in mouse line using CRISPR/Cas9.
(A) A schematic overview of the strategy for generating the PdgfrartTA knock-in allele. The gRNA coding sequence is as shown, and the protospacer-adjacent motif (PAM) sequence is indicated in green. …
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Figure 1—source data 1
Raw data for Figure 1.
- https://doi.org/10.7554/eLife.36865.005
Figure 1—figure supplement 1
PCR confirmation of the correct homologous recombination following CRISPR/Cas9 injection and the labeling efficiency of PdgfrartTA mice.
(A–B) Primer sets LF, LB and RF, RB as indicated in Figure 1A were used in genomic PCR on tail DNA of CRISPR/Cas9 injected founder animals. Two samples showed positive homologous …
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Figure 1—figure supplement 1—source data 1
Raw data for Figure 1—figure supplement 1.
- https://doi.org/10.7554/eLife.36865.004
Figure 2 with 3 supplements
Lineage tracing of Pdgfra cells starting prenatally.
(A) Timeline of the start of dox treatment and analysis. (B–M) Representative immunofluorescent staining of P7 PdgfrartTA;tetO-cre;Rosa-tdTomato lungs. Markers are as indicated: tdTomato (red), the …
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Figure 2—source data 1
Raw data for Figure 2.
- https://doi.org/10.7554/eLife.36865.011
Figure 2—figure supplement 1
Tracing of Pdgfra-lineaged cells in prenatal stage.
(A) Representative immunofluorescent staining for tdTomato (red) and DAPI (blue) in lungs of PDGFRartTA;tetO-cre;Rosa-tdTomato mice at P7 without dox feeding. (B,B’) Representative immunofluorescent …
Figure 2—figure supplement 2
Tracing of Pdgfra-lineaged cells in the early postnatal stage.
(A) Timeline of dox treatment and analysis. PdgfrartTA;tetO-cre;Rosa-tdTomato mice were given one dose of dox injection at P0 followed by dox food until analysis at P7. (B–M): Representative …
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Figure 2—figure supplement 2—source data 1
Raw data for Figure 2—figure supplement 2
- https://doi.org/10.7554/eLife.36865.009
Figure 2—figure supplement 3
Lineage tracing of PDGFRa cells to adult.
(A) Timeline of dox treatments and analysis. (B–G) Representative immunofluorescent staining of PdgfrartTA;tetO-cre;Rosa-tdTomato mice at P40. Markers include: tdTomato (red), SM22a (green) and DAPI …
Figure 3 with 1 supplement
Single cell transcriptome analysis of Pdgfra+ cells during alveologenesis.
(A, D) tSNE plots of scRNAseq data from cells isolated from P7 (A) and P15 (D) PdgfraGFP lungs. Cells can be clustered into five and four main distinct populations at P7 and P15, respectively. …
Figure 3—figure supplement 1
FACS sorting of GFP+ cells from P15 PdgfraGFP mice.
Representative FACS plot showing GFP+ cells in P15 PdgfraGFP mice.
Figure 4
Genetic ablation of Pdgfrα+ cells led to simplified alveoli.
(A) Timeline of dox treatments and analysis. (B) qRT-PCR analysis indicated decreased Pdgfra expression in PdgfrartTA;tetO-cre;Rosa-Dta lungs compared to controls (1 ± 0.06 for control and 0.42 ± 0.0…
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Figure 4—source data 1
Raw data for Figure 4.
- https://doi.org/10.7554/eLife.36865.015
Figure 5 with 1 supplement
Pdgfra-lineage cells increased and contributed to myofibroblasts following bleomycin-induced injury.
(A) Timeline of dox treatment, bleomycin administration and analysis. PdgfrartTA;tetO-cre;Rosa-tdTomato mice were given one dose of dox injection 7 days (D) before bleomycin injury. Lungs were …
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Figure 5—source data 1
Raw data for Figure 5.
- https://doi.org/10.7554/eLife.36865.018
Figure 5—figure supplement 1
Pdgfra-lineaged cells proliferate following bleomycin treatment.
(A–D, A’–D’) Representative immunofluorescent staining for tdTomato (red), Ki67 (green) and DAPI (blue) in lungs of PBS-treated controls and bleomycin-treated mice (B, B’) on D14 or D21 post …
Figure 6
Pdgfra-lineaged cells decrease following neonatal hyperoxia-induced injury.
(A) Timeline of dox and hyperoxia treatment. Pregnant females were fed dox food between E9.5 and P0. Pups and mother were then fed food without dox, raised in 75% O2 or room air from P0 to P12 and …
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Figure 6—source data 1
Raw data for Figure 6.
- https://doi.org/10.7554/eLife.36865.020
Figure 7
A model illustrating the lineage potential of Pdgfra-lineaged cells in development and pathogenesis.
In normal development (black notes), Pdgfra-lineaged cells give rise primarily to myofibroblasts, and minimally to lipofibroblasts and endothelial cells. In pathogenesis (blue notes), Pdgfra-lineaged…
Tables
Key resources table
| REAGENT or RESOURCE | Designation | Source | Identifier | Additional information |
|---|---|---|---|---|
| strain, strain background (mouse) | tetO-GFP, mix background | Jackson Laboratories | 016836 | |
| strain, strain background (mouse) | PdgfraGFP , mix background | Jackson Laboratories | 007669 | |
| strain, strain background (mouse) | tetO-Cre, B6 background | Jackson Laboratories | 006234 | |
| strain, strain background (mouse) | Rosa-tdTomato, B6 background | Jackson Laboratories | 007914 | |
| strain, strain background (mouse) | Rosa-Dta, mix background | Jackson Laboratories | 006331 | |
| Antibody | Elastin | Abcam | ab21600, 1:500 | |
| Antibody | SM22a | Abcam | ab14106, 1:200 | |
| Antibody | ADRP | Abcam | ab52356, 1:200 | |
| Antibody | ERG | Abcam | ab92513, 1:200 | |
| Antibody | ICAM2 | BD | 553325, 1:200 | |
| Antibody | Ki67 | Abcam | ab15580, 1:200 | |
| Antibody | a-SMA | Sigma | F3777, 1:200 | |
| Antibody | goat anti rabbit FITC | Jackson Immuno Research Laboratories | 111-095-144, 1:200 | |
| Antibody | goat anti rabbit Cy3 | Jackson Immuno Research Laboratories | 111-165-144, 1:200 | |
| Antibody | goat anti rat FITC | Jackson Immuno Research Laboratories | 112-095-003 1:200 |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.36865.022
