PI3K signaling specifies proximal-distal fate by driving a developmental gene regulatory network in SOX9+ mouse lung progenitors
- Department of Pediatrics, University of Cincinnati, United States
- Perinatal Institute, Cincinnati Children's Hospital Medical Center, United States
- Division of Neonatology, Perinatal and Pulmonary Biology, Cincinnati Children's Hospital Medical Center, United States
- Wake Forest University, United States
- Department of Medicine, University of Cincinnati, United States
Figures
Figure 1 with 1 supplement
The SOX9+multi-potent lung epithelial progenitor cell population.
(A–C) Whole mount confocal imaging of embryonic lungs isolated from SOX9-GFP reporter mice at E12.5 (A), E13.5 (B), and E14.5 (C) show SOX9 expression at the distal branch tips and in the tracheal …
Figure 1—figure supplement 1
Single-cell analysis of mouse embryonic lung.
(A) Sample batches are indicated (red – E11.5 lung epithelium; blue – E16.5 lung epithelium). (B) Monocle pseudotime heatmap is shown. (C–D) Gene enrichment modules 4 and 12 are highly enriched for …
Figure 2 with 4 supplements
The SOX9+progenitor cell chromatin accessibility landscape.
(A) Schematic strategy for isolation of SOX9+EPC. Cells sorted at E11.5 and E16.5 were used for RNA-seq and ATAC-seq analysis (n=2 biological replicates pooled from 8 to 12 embryos at each timepoint …
Figure 2—figure supplement 1
Bulk RNA-Seq analysis of the developing SOX9+EPC population.
(A) A Volcano plot for genes differentially expressed between E11.5 and E16.5 in SOX9+EPCs is shown. (B–C) Gene ontology analysis demonstrates that differentially expressed genes at E11.5 are …
Figure 2—figure supplement 2
FACS sorting strategy for isolation of Sox9+EPC cells.
SOX9+lung epithelial progenitor cells were isolated by cell sorting CD326+/CD31-/CD45-/7-AAD-/GFP + cells isolated from Sox9-GFP reporter embryos at E11.5 and E16.5. Key single stain controls are …
Figure 2—figure supplement 3
ATAC-Seq peak characteristics.
(A) A representative locus for a well-expressed gene (Nkx2.1) shows the high degree of consistency between biological replicates, and strong correlation between open regions of chromatin with …
Figure 2—figure supplement 4
Accessible chromatin regions, associated histone marks, and neighboring gene expression.
(A–B) The relationship between gene expression and the presence of a nearby region of accessible chromatin is displayed. Data is shown as the odds that a gene is expressed, compared to the set of …
Figure 3
Paired expression and chromatin acessibility modeling of SOX9+progenitor cells.
(A) The computational model Paired Expression and Chromatin Accessibility (PECA) was used to develop a SOX9+ epithelial progenitor cell gene regulatory network. Potential active cis-regulatory …
Figure 4
PI3K signaling in ATAC data.
(A–C) Gene ontology analysis for genes nearest E11.5 and E16.5 differentially accessible regions (DARs) and common regions are shown. Pathways regulating lung epithelial development, such as Wnt, …
Figure 5
Phospho-AKT staining in the developing lung epithelium.
(A–F) Strong staining for AKT phosphorylated at Ser473 (pAKT) was observed in the early lung epithelium at E12.5, with minimal staining seen in the mesenchyme. Staining intensity was highest in the …
Figure 6 with 1 supplement
Conditional deletion of Pik3ca from the developing lung epithelium results in impaired branching morphogenesis and cystic pulmonary hypoplasia.
(A–B) Whole-mount images show cystic areas throughout the lungs of Pik3ca cKO embryos at E18.5. The lungs are smaller in size compared to littermate controls. (C–F) Widefield imaging of H&E stains …
Figure 6—figure supplement 1
Conditional deletion of Pik3ca with Shh-Cre results in loss of epithelial pAKT staining.
(A–C) In littermate control embryos, immunofluorescence microscopy clearly detected phospho-AKT (pAKT) in the lung epithelium at E12.5. (D–F) In Pik3ca cKO embryos, no detectable pAKT signal was …
Figure 7
Loss of Pik3ca in the developing lung epithelium leads to persistence of the SOX9+epithelial progenitor cell population at E18.5.
(A–AF) The relative numbers of NKX2−1+epithelial cells, SOX9+ epithelial progenitor cells, and fraction of proliferating (Ki67+) cells within each population were assessed and quantified using …
Figure 8
Loss of Pik3ca in the developing lung epithelium leads to impaired airway epithelial cell differentiation.
(A–X) SOX2 expression is observed in the airway epithelium from E12.5 to E18.5. (NN) Although the total number of SOX2 + cells present at E18.5 appears to be decreased in images A-X, the relative …
Figure 9
A model of the role of PI3K signaling in the developing lung epithelium.
(A) During normal lung epithelial development, a proximal-to-distal gradient of PI3K signaling patterns the lung epithelium, with highest levels in the developing conducting airways and distal tips …
Author response image 1
Additional files
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Supplementary file 1
Genes identified to be differentially expressed between E11.5 and E16.5 in SOX9+lung epithelial cells sorted using flow cytometry are listed.
- https://cdn.elifesciences.org/articles/67954/elife-67954-supp1-v2.xlsx
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Supplementary file 2
Significantly accessible regions identified using MACS2 in SOX9+lung epithelial cells sorted using flow cytometry at E11.5 are listed.
- https://cdn.elifesciences.org/articles/67954/elife-67954-supp2-v2.xlsx
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Supplementary file 3
Significantly accessible regions identified using MACS2 in SOX9+lung epithelial cells sorted using flow cytometry at E16.5 are listed.
- https://cdn.elifesciences.org/articles/67954/elife-67954-supp3-v2.xlsx
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Supplementary file 4
Regions of differentially accessible chromatin in SOX9+lung epithelial cells sorted using flow cytometry, with increased accessibility at E11.5 compared to E16.5, are listed.
- https://cdn.elifesciences.org/articles/67954/elife-67954-supp4-v2.xlsx
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Supplementary file 5
Regions of differentially accessible chromatin in SOX9+lung epithelial cells sorted using flow cytometry, with increased accessibility at E16.5 compared to E11.5, are listed.
- https://cdn.elifesciences.org/articles/67954/elife-67954-supp5-v2.xlsx
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Supplementary file 6
Regions of accessible chromatin in SOX9+lung epithelial cells sorted using flow cytometry, that did not undergo statistically significant changes in accessibility between E11.5 and E16.5 (common regions), are listed.
- https://cdn.elifesciences.org/articles/67954/elife-67954-supp6-v2.xlsx
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Supplementary file 7
Information for ENCODE datasets used in this study are listed.
- https://cdn.elifesciences.org/articles/67954/elife-67954-supp7-v2.xlsx
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Supplementary file 8
Oligonuclotide DNA sequences used as primers for RT-PCR (qPCR) in this study are listed.
- https://cdn.elifesciences.org/articles/67954/elife-67954-supp8-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/67954/elife-67954-transrepform1-v2.pdf
