Supplemental data of Glucocorticoid Dependent Mesenchymal Cell Differentiation is Required for Perinatal Lung Morphogenesis and Lung Function
Creators: name: Xu, Yan
name: Parvathi Sudha
name: Yina Du
Publisher: name: figshare
Year: 2020
Subjects: subject: 60102 Bioinformatics
subject: FOS: Computer and information sciences
subject: FOS: Computer and information sciences
subject: Computational Biology
subject: 60405 Gene Expression (incl. Microarray and other genome-wide approaches)
subject: FOS: Biological sciences
subject: FOS: Biological sciences

description: <b>Supplemental Figure S1: Single cell transcriptomic mapping of embryonic pulmonary cells.</b> Single cell RNA-seq of fetal mouse lung at E16.5 and E18.5 were generated previously by Fluidigm C1. Cell type mapping and specific signature genes are available in LGEA web portal. A) Plot depicts a two-dimensional representation (tSNE) of single cell RNA profiles of cells isolated from E16.5 lung. Distinct cell types were identified including epithelial, endothelial, pericytes, myeloid/immune, proliferative mesenchymal progenitor (PMP), myofibroblast/smooth muscle (MyoFB), matrix fibroblast (MFB), and two intermediate fibroblast cell types (IF1 and IF2 cells). B) tSNE of single cell RNA profiles of cells isolated from E18.5 lung shows distinct cell types including alveolar type I (AT1) and type II (AT2), ciliated, club, endothelial, myeloid/immune, lipo/matrix fibroblasts and myofibroblast/smooth muscle cells. C, D) E16.5 epithelial cells were further dissected into four subtypes via principle components analysis: Sox9+ progenitors (red), Foxa2+ progenitors (green), pre-AT1 cells (blue) and pre-AT2 cells (orange). Top 300 most variable genes were used to PCA plots. Genes are displayed in four different color markers to represent four subtypes. PCA biplots (principle components 1-3) projects the sub-cluster of genes and cells into different sub-spaces based on matrix correlation.<b> </b><b>Supplemental Figure S2. Lung sacculation defect in mice with mesenchymal deletion of glucocorticoid receptor<i>. </i></b>A) H&amp;E stained lung sections from E18.5 control (panels A-C) or littermate Twist2Cre:Nr3c1fl/fl mice (panels D-F). Note decreased airspaces in Twist2Cre:Nr3c1fl/fl lungs as compared to littermate controls. B) Quantification of airspace area in E18.5 controls and Twist2Cre:Nr3c1fl/fl mice. Scale bars = 100um. Each data point represents an individual animal.<b> </b><b>Supplemental Figure S3. Immunofluorescence staining of CD106, FN1 and ACTA2 protein in control E18.5 lung tissue. </b>CD106 expression is restricted to the mesenchyme, similar to vimentin. Note lack of CD106 co-expression with SMA and lack of CD106 staining in proximal (solid arrow) and distal (open arrow) epithelial cells. Scale bar = 40um.<b>Supplemental Figure S4: </b><b>Expression of cell type selective markers in CD106+ vs. CD106- cells.</b> Expression of cell type selective markers in CD106+ (blue bars) vs CD106- (orange bars) cells. Marker genes for matrix fibroblasts (MFB, red), proliferative mesenchymal progenitors (PMP, dark blue), myofibroblasts/smooth muscle (MyoFB/SM, violet), endothelial (Endo, green), and epithelial cells (Epi, light blue) are shown on the X-axis. Y-axis represents normalized RNA expression (FPKM), Mean±SEM. CD106+ cells express high levels of MFB markers; low levels of epithelial markers were also detected as the population was highly enriched but not 100% pure. 80-100k cells were analyzed per sample.<b>Supplemental Figure S5: Functional enrichment analysis of genes with ATAC-seq open chromatin peaks at E16.5 and E18.5.</b> CD106+ cells were isolated from normal mouse fetal lungs at E16.5 and E18.5 using negative selection (CD45/CD31/CD309) followed by positive selection of CD106. Cells were treated with 100nM Dex or saline for 4 hours in vitro and subjected to ATACseq. Cells were pelleted, counted and 50,000 cells per sample prepared for ATAC-seq at a depth of ~1,000,000 reads per sample. ATAC-seq peaks with more than 2 fold enrichment in Dex treated vs. saline controls at E16.5 (blue) and E18.5 (orange) were selected and nearby genes were annotated based on mm10 genome using Homer. Those genes were subject to functional enrichment analysis using ToppGene suite. Shared functional enrichment by Dex treatment were observed at both time points. The transcription factor notations reflect the type of position-specific scoring matrix used (e.g., V$ for vertebrate matrix library).<b>Supplemental Figure S6: NR3C1 centered transcription regulatory network.</b> Genes differentially expressed in lung mesenchymal cells from GR gain (Dex treated MFB) and loss-of-function ( (Col1a2Cre;Nr3c1<sup>fl/fl</sup>) at E18.5 using were subject to IPA (Ingenuity) Analysis to identify potential upstream regulators of these altered genes. Common key regulators for genes changed in both GR gain- and loss-of-function conditions were predicted and depicted in the NR3C1 centered network (blue nodes). Genes induced in Dex treated MFB cells are shown in green and genes decreased in Nr3c1 mesenchymal deletion lungs are shown in red. The biological relationship between regulators and differential expressed genes are represented by an edge (line). The network was generated in Cytoscape v3.7.1. Due to the space limit, altered genes are invisible in green or red nodes, complete differentially expressed gene lists are provided in supplementary tables. As shown, differentially expressed genes in response to GR gain and loss-of-function are likely regulated by a same group of upstream regulators (i.e., shared same regulatory mechanism mediated by GR). <b>Supplemental Figure S7</b>: <b>Predicted GC/GR dependent mesenchymal-epithelial ligand-Receptor interactions. </b>A) Predicted GC/GR dependent ligand-Receptor interactions via paracrine (MFB to EPI) or autocrine (MFB-MFB). We first identified ligands expressed in PMP and MFB lineages and linked them with their known receptors present in both mesenchyme and epithelium to predict autocrine and paracrine signaling pathways related to GR. Red and green nodes represent ligands increased or decreased in mesenchymal GR deletion lung (Col1a2Cre;Nr3c1<sup>fl/fl</sup>) at E18.5, respectively. Ligand/Receptor interactions were collected from DLRP, KEGG and FANTOM5 project. B) Representative ligands and receptors that were altered after mesenchymal GR deletion and/or by Dex treatment prior to birth.<b></b> <b>Supplemental Table S1: E16.5 differentially expressed genes from Microarray</b><b>Supplemental Table S2: E18.5 differentially expressed genes from Microarray</b><b>Supplemental Table S3: Motif enrichment analysis results of ATAC-seq samples</b> <b>Supplemental Table S4: Fisher's exact test on occurrence of the known GR targets in ATAC-seq samples</b>