Fig 1.
Immunophenotypic characterization of pulmonary lymphatic endothelium at E16.5 and E18.5.
(A) Prox1 (white nuclei) in fetal lungs colocalized with Vegfr3 (green) and the pan-endothelial marker CD31 (red). (B) Prox1 (white) also colocalized with Pdpn (green) and Lyve1 (red) at E16.5 and E18.5. Colocalization of Vegfr3, CD31, Pdpn and Lyve1 staining was observed only in pulmonary lymphatic endothelium as indicated by white arrows (scale bar, 100 um).
Fig 2.
Isolation and relative purity of sorted CD45-/Epcam-/ CD31+/Vegfr3+/Lyve1+/Pdpn+ PLECs.
(A, left) Cell sorting algorithm depicting three major cell populations; CD31-/Vegfr3- (grey), CD31+/Vegfr3- (yellow), CD31+/Vegfr3+ (purple) observed after excluding CD45+, Epcam+ and dead cells (E18.5 shown). (A, right) Expression of Lyve1 and Pdpn in each of the three cell fractions. (B) qRT-PCR measurements of relative Prox1, Flt4/Vegfr3, and Emcn expression in each sorted lung fraction. Statistical significance was determined using a two-tailed Student’s t test (***p<0.001, **p<0.01, *p<0.05; n = 3 per cell fraction).
Fig 3.
Enrichment of lymphatic markers in sorted PLECs.
qRT-PCR measurement of the lymphatic markers, Prox1, Flt4/Vegfr3, and Lyve1 in sorted E18.5 PLECs (n = 4) versus E18.5 whole lung (n = 3). Statistical significance was determined using an unpaired two-tailed Student’s t test (***p<0.001).
Fig 4.
Step-wise gating strategy for isolation of PLECs (CD45-/Epcam-/CD31+/Vegfr3+/Lyve1+/Pdpn+) from late-gestation fetal lungs.
Representative gate for live, CD45- and Epcam- lung cells is boxed (left panels). The gate containing the CD45-/Epcam-/CD31+/Vegfr3+ population (boxed, middle panels). (Right panels) The CD45-/Epcam-/CD31+/Vegfr3+ cellular fraction expressed both Pdpn+ and Lyve1+ characteristic of the lymphatic endothelial phenotype across late-gestation (boxed, right panels).
Fig 5.
Temporal-dependent gene expression in fetal PLECs.
(A) Principal component analysis of E16.5, E17.5, and E18.5 sorted PLECs (n = 3 per time point). Each axis indicates the fraction of experimental variance explained by each principal component (PC). (B) Heatmap of all 1,281 genes with significant differential expression with respect to time (ANOVA FDR q < 0.05). The number of each cluster is indicated at the bottom of the heatmap. Expression values for each gene were z-normalized to a mean of zero and standard deviation of one across all samples in each row; blue, white, and red indicate final z-scores of ≤ -2, 0, and ≥ 2, respectively.
Table 1.
Classification of genes upregulated in E18.5 vs. E16.5 PLECs (FC≥2, p<0.05, FDR q<0.25).
Table 2.
Classification of genes downregulated in E18.5 vs. E16.5 PLECs (FC≤-2, p<0.05, FDR q<0.25).
Fig 6.
Leading edge heatmaps of selected GSEA results.
The expression of the leading edge genes of each GSEA result is shown for (A) Reactome "interferon α/β signaling", (B) KEGG "complement and coagulation cascades", (C) Gene Ontology "regulation of angiogenesis", and (D) Reactome "DNA replication". Genes (rows) are arranged in descending order by the magnitude of the E18.5 vs E16.5 t statistic. Expression values for each gene were z-normalized to a mean of zero and standard deviation of one across all samples in each row; blue, white, and red indicate final z-scores of ≤ -2, 0, and ≥ 2, respectively.
Fig 7.
Down-regulation of cell-cycle and upregulation of IFN pathway genes in fetal PLECs.
qRT-PCR analysis of gene expression in independently sorted PLEC samples, (E16.5, n = 3; E18.5, n = 2 or n = 3 for Ifit1 and Irf7). Statistical analysis was performed using an unpaired 1-tailed Student t test (**p<0.01, *p<0.05).
Fig 8.
Increased expression of Ifit1 mRNA in E18.5 PLECs in vivo.
Single molecule fluorescent in situ hybridization for Ifit1 mRNA (red punctae) and Vegfr3 (green) immunostaining in E16.5 and E18.5 lungs. Boxed, numbered insets have been cropped, magnified and shown at the right of each image. Note the increased red punctae representing Ifit1 mRNA in Vegfr3+ PLECs at E18.5. (scale bar, 50 um).
Fig 9.
PLEC-specific transcriptional events during fetal lung maturation.
Gene Set Enrichment Analysis (GSEA) demonstrated that genes annotated with the GO term "chemokine activity" (GO:0008009) (A) or NF-κB target genes (TRANSFAC motif V$NFKAPPAB_01) (B) were significantly (FDR q < 0.25) coordinately upregulated during fetal maturation in PLECs but not in whole lung (GEO Series GSE35485). Expression of the genes in the leading edge of each PLEC GSEA result is shown in both datasets, with red and blue indicating values up- or down-regulated, respectively, at least 3-fold from the mean (white) across all samples within each dataset (A-B). (C) The expression of select genes changes significantly (one-way ANOVA p < 0.05) with respect to time in sorted PLECs (upregulated from E16.5 to E18.5) but not in the whole lung.