Supplemental data of Glucocorticoid Dependent Mesenchymal Cell Differentiation is Required for Perinatal Lung Morphogenesis and Lung Function
Creators: name: Xu, Yan
name: Parvathi Sudha
Publisher: name: figshare
Year: 2019
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>Supplemental Figure S2. Lung sacculation defect in mice with mesenchymal deletion of glucocorticoid receptor.</b> A) H&amp;E stained lung sections from E18.5 control (panels A-C) or littermate Twist2Cre:Nr3c1<sup>fl/fl</sup> mice. Note decreased airspaces in Twist2Cre:Nr3c1<sup>fl/fl</sup> lungs. B) Quantification of airspace areas in E18.5 controls and Twist2Cre:Nr3c1<sup>fl/fl</sup> mice demonstrates decreased sacculation in GR deletion mice at E18.5. <b>Supplemental Figure S3. Identification of distinct mesenchymal cell types and isolation of matrix fibroblast cells (MFB).</b> A) A subset of lung fibroblasts, MFBs, were identified by CD106 staining at E16.5 in wild type mice. B) CD106+ cells were isolated from E16.5 wild type lungs after negative depletion of immune and endothelial cells (CD45/31/144/309) followed by positive selection with a CD106 antibody (yield ~ 1.5x105 cells per lung). Bulk RNA-seq analysis of CD106+ vs CD106- cells from wild type mice (n=2 isolations/group) demonstrated enrichment of MFB-signature genes in CD106+ cells in comparison with PMP- and MyoFB-signature genes, two additional lung mesenchymal cell subtypes similar to MFBs. Volcano plot shows signature genes from PMP (dark blue), MyoFB (violet), MFB (red), Endo (green) and Epithelium (light blue) that were induced more than 1.5-fold in CD106+ cells. Grey color represents genes that were not cell type specific. C) 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. <b>Supplemental Figure S4: 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. 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. <b>Supplemental Figure S5: NR3C1 centered transcription regulatory network.</b> Upstream analysis was performed on differentially expressed genes in lung mesenchymal cells from GR gain- (MFB cells identified from scRNA-seq analysis of fetal lungs whose dams were treated with Dex in vivo, details see method section) and loss-of-function (lung RNA from mesenchyme-specific GR knockout (Col1a2Cre;Nr3c1<sup>fl/fl</sup>)) conditions at E18.5 using Ingenuity Pathway Analysis. Common key regulators in both GR gain- and loss-of-function conditions were 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. 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 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>