Atf3 defines a population of pulmonary endothelial cells essential for lung regeneration

  1. Terren K Niethamer
  2. Lillian I Levin
  3. Michael P Morley
  4. Apoorva Babu
  5. Su Zhou
  6. Edward E Morrisey  Is a corresponding author
  1. Department of Medicine, United States
  2. Department of Cell and Developmental Biology, United States
  3. Penn-Children’s Hospital of Philadelphia Lung Biology Institute, University of Pennsylvania, United States
  4. Penn Cardiovascular Institute, University of Pennsylvania, United States
7 figures, 1 table and 1 additional file

Figures

Figure 1 with 3 supplements
Expression of Atf3 defines a subset of pulmonary capillary endothelial cells.

(A) The Uniform Manifold Projection (UMAP) of endothelial cell subtypes from single-cell RNA sequencing (scRNA-seq) of whole mouse lung at homeostasis (n=1). (A’, A'') Atf3 transcript expression in …

Figure 1—figure supplement 1
Pulmonary endothelial cell (EC) subclusters at homeostasis are defined by their differentially expressed genes.

(A) Both CAP1_As and CAP1_Bs express general pulmonary capillary EC marker Gpihbp1. (B) The CAP1_B cluster is defined by the expression of transcription factor Atf3. (C) CAP2s express the known …

Figure 1—figure supplement 2
Pulmonary endothelial cell (EC) subclusters at 14 days post-H1N1 are defined by their differentially expressed genes.

(A) CAP1s express marker gene Gpihbp1 across both A and B subclusters. (B) CAP1_Bs are additionally defined by the expression of the transcription factor Atf3. (C) CAP2s are defined by the …

Figure 1—figure supplement 3
Integration of control and influenza single-cell RNA sequencing (scRNA-seq) data.

(A) The Uniform Manifold Projection (UMAP) dimension reduction plots from the integration of control and influenza scRNA-seq data, with colored dots representing cellular contributions to each …

Figure 2 with 3 supplements
Atf3-expressing CAP1_Bs increase in the mouse lung after H1N1 injury and contribute to endothelial regeneration.

(A) Schematic of the experimental setup to investigate Atf3-expressing cells during regeneration after H1N1. Atf3CreERT2; ROSA26LSL-tdTomato (Atf3-tdTm) adult mice (5–13 weeks old) received 200 …

Figure 2—figure supplement 1
Atf3CreERT2 activity is not induced by H1N1 injury.

(A) Atf3CreERT2; ROSA26LSL-tdTomato (Atf3-tdTm) adult mice treated with tamoxifen 8 days after H1N1 influenza injury show robust tdTomato expression at 22 days post-injury (dpi). (B) Atf3-tdTm adult …

Figure 2—figure supplement 2
Atf3 is expressed at low levels in other alveolar cell types, but these cells do not increase in number after H1N1.

(A) Immunostaining for the AT2 cell marker DC-LAMP (LAMP3), red, and the alveolar epithelial type 1 (AT1) cell marker HOPX, green, in Atf3CreERT2; ROSA26LSL-tdTomato adult mice treated with …

Figure 2—figure supplement 3
Atf3-expressing endothelial cells increase in the alveolar space following H1N1 infection.

(A) A tile scan of immunofluorescence (IF) for the nuclear endothelial marker ERG, cytoplasmic endothelial marker Endomucin-1, and tdTomato in Atf3-tdTm animals at homeostasis show that …

Figure 3 with 4 supplements
Endothelial loss of Atf3 causes defects in alveolar regeneration.

(A) Schematic of the experimental setup. Cdh5-CreERT2; ROSA26LSL-tdTomato (Atf3EC-WT) and Cdh5-CreERT2; ROSA26LSL-tdTomato; Atf3lox/lox (Atf3EC-KO) adult mice (5–15 weeks old) received 200 mg/kg …

Figure 3—figure supplement 1
Quantification of the alveolar area using immunohistochemistry for podoplanin (PDPN).

(A) Example 20 X image taken from a lung tissue section from a Cdh5-CreERT2; ROSA26LSL-tdTomato; Atf3lox/lox (Atf3EC-KO) mouse at 90 days post influenza (dpi). Immunofluorescence (IF) was performed …

Figure 3—figure supplement 2
Atf3EC-WT and Atf3EC-KO animals have similar levels of tissue damage after influenza infection.

(A) Representative images of Atf3EC-WT and (B) Atf3EC-KO lung tissue at 21 days post influenza stained with hematoxylin and eosin (H&E) demonstrate areas of tissue damage caused by H1N1 infection in …

Figure 3—figure supplement 3
Endothelial loss of Atf3 at homeostasis does not cause defects in lung tissue structure or cell loss after 28 days.

(A) Schematic of the experimental setup. Cdh5-CreERT2; ROSA26LSL-tdTomato mice (Atf3EC-WT) and Cdh5-CreERT2; ROSA26LSL-tdTomato; Atf3lox/lox (Atf3EC-KO) mice were given tamoxifen at 200 mg/kg by …

Figure 3—figure supplement 4
Loss of endothelial Atf3 at homeostasis does not cause tissue structure defects after 6 months.

(A) Schematic of the experimental setup. Cdh5-CreERT2; ROSA26LSL-tdTomato mice (Atf3EC-WT) and Cdh5-CreERT2; ROSA26LSL-tdTomato; Atf3lox/lox (Atf3EC-KO) mice were given 200 mg/kg tamoxifen by oral …

Figure 4 with 1 supplement
Transcriptional changes due to loss of Atf3 after acute influenza injury.

(A) Schematic of the experimental setup. Cdh5-CreERT2; ROSA26LSL-tdTomato (Atf3EC-WT, n=4) and Cdh5-CreERT2; ROSA26LSL-tdTomato; Atf3lox/lox (Atf3EC-KO, n=3) adult mice received 200 mg/kg tamoxifen …

Figure 4—figure supplement 1
qRT-PCR for expression of Atf3 and downstream genes in Atf3EC-WT and Atf3EC-KO animals.

(A) qRT-PCR for Atf3 demonstrates lower expression in Atf3EC-KO (n=9) compared to Atf3EC-WT (n=8) mouse lung endothelial cells. Genes shown to be downregulated by RNA sequencing also show lower …

Figure 5 with 3 supplements
Endothelial loss of Atf3 causes alveolar endothelial cell loss corresponding to regions of altered alveolar architecture.

(A) Immunofluorescence (IF) for tdTomato (magenta) and DAPI (blue) at 14 days post-H1N1 shows an expected distribution of tdTomato+ ECs throughout the alveolar space in Atf3EC-WT animals. (A’) …

Figure 5—figure supplement 1
Endothelial cell loss in regions of altered alveolar architecture persists at 21 days post-H1N1.

(A) Representative image of lung tissue from an Atf3EC-WT mouse at 21 dpi with immunofluorescence (IF) for (A’) cytoplasmic endothelial marker Endomucin-1 (EndoM1, green), (A’’) tdTomato (magenta), …

Figure 5—figure supplement 2
Endothelial loss of Atf3 leads to loss of alveolar epithelial type 1 (AT1) and AT2 cells but not alteration in AT2-AT1 differentiation.

(A) Representative image from lung tissue section of an Atf3EC-WT mouse at 21 dpi. Immunofluorescence (IF) analysis was performed for the pan-epithelial marker NKX2-1 (white), the AT2 cell marker …

Figure 5—figure supplement 3
Endothelial loss of Atf3 does not impair CAP1-CAP2 differentiation.

(A) Representative image from lung tissue section of an Atf3EC-WT mouse at 21 dpi. RNAscope was performed for the CAP2 marker Car4 (white), and IF analysis was performed for tdTomato (red) and the …

Atf3 loss in endothelial cells (ECs) impairs the regenerative response to H1N1.

(A) Compared to Atf3EC-WT mice (n=4), Atf3EC-KO mice (n=4) have significantly decreased EC proliferation (%CD31+/EdU+ of CD45- cells) between 7–14 dpi. Error bars represent standard deviation (SD). …

Loss of Atf3 in endothelial cells (ECs) impairs long-term alveolar tissue regeneration.

(A) Schematic of the experimental setup. Cdh5-CreERT2; ROSA26LSL-tdTomato (Atf3EC-WT) and Cdh5-CreERT2; ROSA26LSL-tdTomato; Atf3lox/lox (Atf3EC-KO) adult mice received 200 mg/kg tamoxifen by oral …

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Mus musculus, male and female)Atf3tm1.1(cre/ERT2)Msra (Atf3CreERT2)Matt RamerMGI: 6360893PMID:26610346
Strain, strain background (Mus musculus, male and female)C57BL/6-Tg(Cdh5-cre/ERT2)1Rha (Cdh5CreERT2)Taconic13073, MGI: 3848982PMID:19144989
Strain, strain background (Mus musculus, male and female)Gt(ROSA)26Sortm14(CAG-tdTomato)Hze (ROSA26LSL-tdTomato)Jackson Laboratory007914, MGI: 3809524PMID:20023653
Strain, strain background (Mus musculus, male and female)Atf3tm1.1Hai (Atf3lox)Tsonwin HaiMGI: 5547956PMID:23921126
AntibodytdTomato (goat polyclonal)OrigeneAB8181-2001:50
AntibodyRFP (DsRed) (rabbit polyclonal)Rockland600-401-379, RRID:AB_22097511:50
AntibodyERG (mouse monoclonal)Abcamab2143411:100
AntibodyERG (rabbit monoclonal)Abcamab92513
RRID:AB_2630401
1:100
AntibodyEndomucin-1 (goat polyclonal)R&D SystemsAF4666 RRID:AB_21000351:500
AntibodyLAMP3 (rat monoclonal)Novus BiologicalsDDX0191P-100 RRID:AB_28275321:100
AntibodyHOPX (mouse monoclonal)Santa Cruz Biotechnologysc-398703 RRID:AB_26879661:100
AntibodyNKX2-1 (TTF1) (rabbit monoclonal)Abcamab76013 RRID:AB_13107841:50
AntibodyPDPN (mouse monoclonal)Abcamab10288 RRID:AB_2970271:100
AntibodyCD45-PerCP-Cy5.5 (rat monoclonal)Thermo Fisher45-0451-82 RRID:AB_11070021:200
AntibodyCD31-PE-Cy7 (rat monoclonal)Thermo Fisher25-0311-82 RRID:AB_27169491:100
Chemical compound, drugPR8-GP33 H1N1 influenzaDr. E. John WherryPMID:23516357
Chemical compound, drugTamoxifenSigma-AldrichT5648
Commercial assay or kitClick-iT Plus EdU Alexa Fluor 488 Flow Cytometry Assay KitThermo FisherC10632
Commercial assay or kitRNAscope Fluorescent Multiplex Reagent Kit v2ACD323100
Commercial assay or kitPureLink RNA Micro KitThermo Fisher12183–016
Commercial assay or kitBioanalyzer High Sensitivity RNA Pico KitAgilent5067–1513
Commercial assay or kitNEBNext Single Cell/Low Input RNA Library Prep Kit for IlluminaNew England BiolabsE6420
Commercial assay or kitBioanalyzer High Sensitivity DNA KitAgilent5067–4626
OtherRNAscope probe mm-Car4-C3ACD468421-C3Probe to detect Car4 RNA; see Figure 5—figure supplement 3 and Materials and methods: RNAscope analysis
OtherRNAscope probe mm-Pdgfra-C2ACD480661-C2Probe to detect Pdgfra RNA; see Figure 2—figure supplement 2 and Materials and methods: RNAscope analysis

Additional files

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